Reversible pegylated drugs

ABSTRACT

Reversible pegylated drugs are provided by derivatization of free functional groups of the drug selected from amino, hydroxyl, mercapto, phosphate and/or carboxyl with groups sensitive to mild basic conditions such as 9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), to which group a PEG moiety is attached. In these pegylated drugs, the PEG moiety and the drug residue are not linked directly to each other, but rather both residues are linked to different positions of the scaffold Fmoc or FMS structure that is highly sensitive to bases and is removable under physiological conditions. The drugs are preferably drugs containing an amino group, most preferably peptides and proteins of low or medium molecular weight. Similar molecules are provided wherein a protein carrier or another polymer carrier replaces the PEG moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of a continuation-in-partapplication of PCT application No. PCT/IL2004/000321, filed Apr. 8,2004, in which in the US is designated, and claims the benefit of U.S.Provisional Patent Application No. 60/460,816, filed Apr. 8, 2003, theentire contents of each and both these applications being herebyincorporated by reference herein in their entirety as if fully disclosedherein.

FIELD OF THE INVENTION

The present invention relates to reversible pegylation of drugs and topegylated drugs that are slowly converted to the drugs in physiologicalconditions.

Abbreviations: ANP, atrial natriuretic peptide; t-Boc,tert-butyloxycarbonyl; BSA, bovine serum albumin; DCC,N,N′-dicyclohexylcarbodiimide; DCU, N,N′-dicyclohexylurea; DMF,N,N′-dimethylformamide; DTNB, 5,5-dithiobis(2-nitrobenzoic acid); ESMS,electrospray ionization mass spectra; Fmoc, 9-fluorenylmethoxycarbonyl;Fmoc-OSu, Fmoc-N-hydroxysuccinimide ester; FMS,2-sulfo-9-fluorenyl-methoxycarbonyl; GSH, reduced glutathione; hGH,human growth hormone; HOSu, N-hydroxy-succinimide; HPLC,high-performance liquid chromatography; HSA, human serum albumin;HSA-Fmoc-insulin, a conjugate of human serum albumin and insulin; IDDM,insulin-dependent diabetes mellitus; IFN-α2, human interferon-α2;ifnar2-EC, extracellular part of IFN-α2 receptor; MAL-FMS-NHS,N-[2-(maleimido-propionylamino)-7-sulfo-fluoren-9-yl-methoxy-carbonyloxy]succinimide (Precursor8); MAL-FMS-OSu, MAL-FMS-NHS; MIB-NHS, maleimido benzoateN-hydroxysuccinimide ester; NHS, N-hydroxy-succinimide; PBS,phosphate-buffered saline; PEG, polyethylene glycol; PEG₅₀₀₀, 5,000Da-PEG; PEG₄₀ or PEG₄₀₀₀₀, 40,000 Da-branched PEG; PEG₄₀-SH, a40-kDa-branched PEG containing a sulfhydryl moiety; PEG₄₀-OSU,PEG₄₀-N-hydroxysuccinimide ester; SC, subcutaneous; STZ, streptozocin;TCA, trichloroacetic acid; TFA, trifluoroacetic acid; THF,tetrahydrofuran; TNBS, 2,4,6-trinitrobenzenesulfonic acid.

BACKGROUND OF THE INVENTION

Most peptide and protein drugs are short-lived and have often a shortcirculatory half-life in vivo. This is particularly valid fornonglycosylated proteins of a molecular mass less than 50 kDa. The shortlifetime of proteins in vivo is attributed to several factors, includingglomerular filtration in the kidney and proteolysis. Considering thatpeptide and protein drugs are not absorbed orally, prolonged maintenanceof therapeutically active drugs in the circulation is a desirablefeature of obvious clinical importance. Proteins with molecular massesabove ˜60 kDa largely avoid glomerular filtration and are not, for themain part, filtered in the kidney. Therefore they remain in thecirculation longer than smaller proteins.

An attractive strategy for improving clinical properties of smallprotein drugs has come to be known as PEGylation (or pegylation, as usedhereinafter). By this strategy several hydrophilic chains ofpolyethylene glycol (PEG) are covalently linked to the protein in orderto increase its effective molecular mass. Important clinical advantagesare gained by pegylation. For example, life-time in vivo can beprolonged in some instances from minutes to hours, owing to the stericinterference that protects conjugates from proteolysis in vivo and theincrease in molecular mass, which precludes filtration by the kidney.Protein pegylation also decreases immunogenicity, presumably byprotecting conjugates from being recognized as foreign antigens by theimmune system.

In spite of the profound advantages often gained by pegylatingtherapeutic proteins, this technology suffers from a principal drawback.On the one hand, covalently attaching PEG chains to proteins prolongstheir lifetime in vivo, protecting the conjugates from proteolysis andshielding them from the immune system. On the other hand, the stericinterference of the PEG chains often leads to a drastic loss or evenabolish the biological and the pharmacological potencies of the proteinsin the conjugates (Fuertges and Abuchowski, 1990; Katre, 1993; Bailonand Berthold, 1998; Nucci et al., 1991; Delgado et al., 1992; Fung etal., 1997; Reddy, 2000; Veronese, 2001). In principle, this deficiencycan be overcome by introducing the PEG chains via a chemical bond thatis sensitive to hydrolysis, or can be cleaved enzymatically by serumproteases or esterases. Clearly, a consistent rate of hydrolysis iscrucial. A prerequisite condition is therefore that the hydrolysis ofthe PEG chains from the conjugate is to take place at a slow rate, andin a homogenous fashion in vivo.

It would be highly desirable to design PEG derivatives of proteins orpeptides or small drug molecules from which PEG can be released byhydrolysis. An appropriate reversible PEG conjugate would have to behydrolyzed slowly and spontaneously in physiological conditions andwould permit time-dependent reactivation of inactive pegylated proteinsand peptides.

Several methods for reversible pegylation were proposed (Greenwald etal., 1999, 2000; Lee et al., 2001; Garman and Kalindjian, 1987; Zalipskyet al., 1999). They suffer, however, from major potential drawbacks. Forexample, reliance on enzymatic detachment as a rate-determining step(Greenwald et al., 1999, 2000; Lee et al., 2001) of PEGs from conjugatesby serum proteases and/or esterases might not yield desirablepharmacokinetic profiles in situ. Moreover, it is dependent on enzymesavailability. Disulfide-bonded conjugate is not to be cleaved in thenon-reducing environment of the body fluids (Zalipsky et al., 1999). Areversibly pegylated conjugate which still retain an active moietycapable of reacting with free SH functions may result in complexundesired cross-linking (Garman and Kalindjian, 1987). It would be verydesirable to design a version of reversible pegylation that wouldovercome these deficiencies.

International PCT Publication No. WO 98/05361 of the present applicantsdescribes a novel conceptual approach for prolonging the half-life ofdrugs by derivatizing a drug having at least one free amino, carboxyl,hydroxyl and/or mercapto groups with a moiety that is highly sensitiveto bases and is removable under mild basic conditions. The prodrugobtained is inactive but undergoes transformation into the active drugunder physiological conditions in the body. Examples of said moietiesare the radicals 9-fluorenylmethoxycarbonyl (Fmoc) and2-sulfo-9-fluorenylmethoxycarbonyl (FMS). According to this concept,Fmoc and FMS derivatives of peptidic drugs such as insulin and humangrowth hormone as well as of non-peptidic drugs such as propanolol,cephalexin and piperacillin have been described in said WO 98/05361.Later on, FMS derivatives of cytokines have been disclosed in WO02/36067, and FMS derivatives of enkephalin, doxorubicin, amphotericinB, gentamicin and gonadotropin releasing hormone (GnRH) were disclosedin WO 02/7859.

U.S. Pat. No. 6,433,135 discloses a pegylated derivative of an analogueof luteinizing hormone releasing hormone (LHRH or GnRH) in which the PEGmoiety is covalently bound to a serine residue of said LHRH analogue. Inthe process of preparation of said PEG-LHRH analogue by solid phasepeptide synthesis, a pegylated serine residue such as Fmoc-Ser(PEG)-QHor tBoc-Ser(PEG)-OH is introduced into the LHRH analogue, and theproduced PEG-LHRH analogue is recovered (without the protective groupFmoc or t-Boc).

JP Patent Application JP 3148298 describes PEG-peptide conjugates, e.g.,PEG-GnRH conjugate, obtained by reacting the guanidino group of anarginine residue with PEG, while protecting the amino groups present inthe molecules.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicants at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

It has been found, in accordance with the present invention, that drugswith a prolonged circulating half-life can be obtained by combining thetechnology of derivatization of the drug with Fmoc or FMS or similarmoieties removable under mild basic conditions with the technology ofattaching a suitable natural or synthetic carrier to the thusderivatized drug molecule, such carrier serving for delivery of the drugand providing further benefits.

The carrier may be a protein such as albumin or a protein containing aglobin-like domain or a polymeric carrier consisting of a biocompatibleand biodegradable polymer containing functional groups. The polymericcarrier is preferably in the form of nanoparticles or it is attached toliposomes.

In one preferred embodiment, the polymeric carrier is PEG. It has beenfound, in accordance with the present invention, that by combination ofthe protein-pegylation technology with the technology of derivatizationwith Fmoc or FMS or similar moieties removable under mild basicconditions, major deficiencies of the protein-pegylation technology,mainly the loss of biological and pharmacological potencies in the PEGconjugates in vivo, may be overcome.

In one embodiment of the present invention, PEG-protein conjugates areprovided from which PEG can be released by hydrolysis underphysiological conditions in the body.

In another embodiment, reversible PEG-protein conjugates are providedthat are inactive when administered and permit time-dependentreactivation of the inactivated pegylated protein under physiologicalconditions in the body.

The present invention thus relates, in one aspect, to a compound of theformula:

(X)_(n)—Y

wherein

Y is a moiety of a drug bearing at least one functional group selectedfrom free amino, carboxyl, phosphate, hydroxyl and/or mercapto, and

X is a radical that is highly sensitive to bases and is removable undermild basic conditions, said radical carrying a protein or a polymericcarrier moiety,

n is an integer of at least one, and pharmaceutically acceptable saltsthereof.

The prodrug obtained is inactive but undergoes transformation into theactive drug Y under physiological conditions in the body.

In preferred embodiments of the invention, the radical X is Fmoc or2-sulfo-Fmoc (herein “FMS”), Y is a peptide or protein drug linked to Ythrough an amino group, n is 1 or 2, the protein carrier is albumin andthe polymeric carrier is a linear or branched PEG moiety having amolecular weight of 5,000-40,000 Da.

In another aspect, the present invention provides novel methods andintermediates and precursors for the preparation of the conjugates ofthe invention.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and aprodrug of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the stability of the maleimide functional moiety inMAL-FMS-NHS in aqueous solutions having different pH values. MAL-FMS-NHS(1 mM) was incubated at room temperature in H₂O (pH 6.0), in 0.007 Macetic acid (pH˜4.0), in 0.1 M phosphate buffer (pH 7.4), and in 0.1 MNaHCO₃ (pH 8.5). At the indicated time points, aliquots were allowed toreact with a slight excess of GSH (15 min at pH 7.2) and theconcentration of unreacted GSH was determined with5,5-dithiobis(2-nitrobenzoic acid) (DTNB).

FIG. 2 shows the degree of incorporation of MAL-FMS-NHS at pH 7.2 intoα-lactalbumin (α-LA) as a function of the amount of added reagent. Tosamples of α-LA (1.0 ml of 1 mg/ml in 0.1M phosphate buffer, pH 7.2),MAL-FMS-NHS was added at concentrations ranging from 1 equivalent up to14 molar equivalents of MAL-FMS-NHS. For each treatment the amountincorporated into the protein was determined by the absorbance at 280nm, after dialysis, and by quantitating the amount of unmodifiedamino-side chain moieties with trinitrobenzene sulfonic acid. (TNBS).

FIGS. 3A-3B show the time course of reactivation of(PEG₅₀₀₀-Fmoc)-2-gentamicin and (PEG₅₀₀₀-Fmoc)₁-gentamicin conjugates,respectively. After incubation at pH 8.5, 37° C., aliquots werewithdrawn at the indicated time points and analyzed for their potency toarrest E. coli replication. The IC₅₀ for each aliquot was determined.Native gentamicin inhibited E. Coli replication with IC₅₀ value0.22±0.02 μM.

FIGS. 4A-4B show progressive modification of the amino acid moieties ofhuman insulin with PEG₅₀₀₀-Fmoc-OSu and loss of biological potency as afunction of PEG₅₀₀₀-Fmoc incorporated into insulin, respectively. (4A)Insulin (17.24 nmoles in 0.2 ml 0.01 M NaHCO₃) reacted with increasingconcentrations of PEG₅₀₀₀-Fmoc-OSu at a molar excess over the protein asindicated in the figure for 2 hours at 25° C. The number of free aminogroups that remained unmodified were quantitated with TNBS. (4B)Aliquots containing 0.4, 0.7, 1.1, 1.5 and 2.2 moles PEG₅₀₀₀-Fmoccovalently attached per mole insulin, were assayed for their lipogenicpotency in rat adipocytes. Under the assay conditions, human insulinstimulates lipogenesis, 4-6 times above basal levels with ED₅₀ value of0.2±0.02 ng/ml. An insulin derivative exhibiting ED₅₀ of 2.0%0.2 ng/mlin this assay is considered as having 10% the lipogenic potency ofnative insulin.

FIG. 5 shows the rate of reactivation of PEG₅₀₀₀-Fmoc-insulin conjugatesupon incubation at pH 8.5, 37° C. PEG₅₀₀₀-Fmoc-insulin conjugatescontaining one and two moles of PEG₅₀₀₀-Fmoc/mole insulin were incubatedat a concentration of 0.172 μM in 0.1 M NaHCO₃-0.5% bovine serum albuminand 1 mM NaN₃ at 37° C. At the indicated time points aliquots wereanalyzed (in several concentrations for each aliquot) for theirlipogenic potencies in rat adipocytes.

FIG. 6 shows prolonged glucose-lowering effect after a singlesubcutaneous (SC) administration of (PEG₅₀₀₀-Fmoc)₁-insulin in mice.Mice received SC, either native insulin (Zn²⁺-free, 1.72 nmole/mouse in0.2 ml PBS buffer) or (PEG₅₀₀₀-Fmoc)₁-insulin (17.2 nmole/mouse, in 0.2ml PBS buffer). Blood glucose levels were determined at the indicatedtime points. Each point is the arithmetic mean±SEM of blood glucose offive mice.

FIG. 7 shows glucose-lowering pattern in mice following singleintraperitoneal (IP) administration of (PEG₅₀₀₀-Fmoc)₁-insulin. Groupsof mice received IP either insulin (Zn²⁺-free, 0.345 nmol/mouse, in 0.2ml PBS buffer) or (PEG₅₀₀₀-Fmoc)₁-insulin (3.45 nmoles/mouse, in 0.2 mlPBS buffer). Blood glucose levels were determined at the indicated timepoints. Each point in the figure is the arithmetic mean±SEM of fivemice.

FIG. 8 shows the rate of release of exendin-4 from PEG₄O-FMS-exendin-4conjugate, upon incubation at pH 8.5, 37° C. At the indicated timepoints, aliquots (50 μl) were loaded on HPLC, and ran under conditionsresolving well exendin-4 from the conjugate. Results are expressed aspercent of maximal peak area of released exendin-4, as a function oftime. Exendin-4 (50 μg) was assigned at 100% peak area.

FIG. 9 shows the rate of hydrolysis of PEG-FMS conjugates, uponincubation at pH 8.5, 37° C. Solutions of PEG₅₀₀₀-FMS-exendin-4(circles) and PEG₅₀₀₀-FMS-4-nitro-phenethyl amine (squares) wereincubated in PBS at pH 8.5, 37° C. At the indicated time points,aliquots (50 μl) were analyzed using HPLC on a RP-4 column. Results areexpressed as percent of the maximal peak area of released exendin-4 and4-nitrophenethylamine, as a function of time.

FIGS. 10A-10B show glucose-lowering patterns of native exendin-4 andPEG₄₀₀₀₀-FMS-exendin-4 following a single subcutaneous administration toCD1-mice. (10A) CD1-mice were SC-administered with either nativeexendin-4 (10 μg/mouse) or with PEG₄₀-FMS-exendin-4 (10 μg/mouse ofexendin-4 equiv). At the indicated time points, circulating glucoselevels were determined. Each experimental group consisted of five mice.Data are presented as means±SE. (10B) Three groups of CD1 mice (n=6 pergroup) underwent one subcutaneous administration of saline, nativeexendin-4 (4 μg/mouse) or PEG₄₀₀₀₀-FMS-exendin-4 (4 μg peptide/mouse).Circulating glucose levels were then monitored. Results are expressed aspercent decrease in plasma glucose concentration in the groups treatedwith exendin-4 or PEG₄₀₀₀₀-FMS-exendin-4 relative to that found in thesaline-treated group measured at the same time-point during the day.

FIGS. 11A-11C show release of active IFNα2 upon incubation ofPEG₄₀-FMS-IFNα2 at pH 8.5, 37° C. PEG₄₀-FMS-IFNα2 (0.3 mg protein/ml)was incubated in 0.1 M phosphate buffer with 2 mM NaN₃ and 6 mg/ml BSA(pH 8.5, 37° C.). At the indicated time points, aliquots were withdrawn.(11A) Analysis of IFNα2 discharge from the conjugate by SDS-PAGE; theamounts of IFNα2 discharge were quantified relative to an IFNα2reference of known concentration and intensity (the time increments andthe percentages are indicated); (11B) Aliquots withdrawn at theindicated time points were analyzed for their Ifnar2 binding capacity onBIAcore; (11C) Fitted BIAcore profile of native IFNα2 discharge fromPEG₄₀-FMS-IFNα2.

FIG. 12 shows the results of SC administration of native IFNα2 andPEG₄₀-FMS-IFNα2. Rats were SC injected with the indicated concentrationsof native IFNα2 (100 μg/rat) or the PEG₄₀-FMS-IFNα2 conjugate (12, 60,120 μg/rat) (0.2 ml/rat, dissolved in PBS). Blood aliquots werewithdrawn at the indicated time points. Circulating antiviral activitiesin the aliquots were determined in human WISH cells with 3-fold serialdilutions of each aliquot.

FIG. 13 shows the result of intravenous administration ofPEG₄₀-FMS-IFNα2 to rats. Rats were intravenously injected with theindicated concentrations of native IFNα2 (30 μg/rat) or thePEG₄₀-FMS-IFNα2 conjugate (30 μg/rat) (0.2 ml/rat, dissolved in PBS).Blood aliquots were withdrawn at the indicated time points. Circulatingantiviral activities in the aliquots were determined in human WISH cellswith 3-fold serial dilutions of each aliquot.

FIGS. 14A-14B show experimental vs. simulated behavior of IFNα2: (14A)following SC administration, with initial concentrations of 60 nM and1.5 nM of PEG₄₀-FMS-IFNα2 and native IFNα2, respectively; (14B)following intravenous administration to rats with initial concentrationsof 20 nM of PEG₄₀-FMS-IFNα2, 1.5 nM of native IFNα2 in the SC volume andno conjugate in circulation. The inserts are the experimental curves.

FIG. 15 shows dose-response of PYY₃₋₃₆ in food intake in mice. MaleC57BL6J mice (10 per group), were deprived of food for 24 h. At time 23h, the mice received a SC injection of either saline or the indicateddoses of PYY₃₋₃₆. At time 24 h the mice were allowed to consume anexcess of pre-weighted chow for 2 h. Drinking water was provided at alltimes. The amount of food consumed per 10 mice during 2 h is shown as afunction of PYY₃₋₃₆ dose.

FIG. 16 shows time-dependent reduction in food intake in mice byPYY_(3-36.)

Male C57BL6J mice (10 per group) were deprived of food as described inFIG. 15 and PYY₃₋₃₆ (5 nmol/mouse) was administered at the indicatedtimes prior to start of the re-feeding period. Results are average offour identical experiments.

FIG. 17 shows the effects of irreversible pegylation on the biologicalactivity of PYY₃₋₃₆. Native PYY₃₋₃₆ was allowed to react with PEG₄₀-OSu.Groups of 10 mice were injected SC with saline, PYY₃₋₃₆, orPEG₄₀-PYY₃₋₃₆ (5 nmol/mouse) at 1 h prior to start of the re-feedingperiod. Results are the average of two identical experiments.

FIG. 18 shows that PEG₄₀-FMS is linked to the α-amino group of PYY₃₋₃₆.PEG₄₀-FMS-PYY₃₋₃₆ (100 μg) was acetylated by a 500 molar excess ofacetic anhydride at pH 7.0, dialyzed, and incubated for 3 days at pH8.5, 37° C. to quantitatively remove the PEG₄₀-FMS moiety. The resultingacetylated PYY₃₋₃₆ was then subjected to three cycles of N-terminalprotein sequence analysis. The sequence obtained wasIle-(NE-acetyl)Lys-Pro. Sequence analysis of the native peptide yieldedIle, Lys, and Pro on cycles 1, 2, 3, respectively (not shown).

FIG. 19 shows the kinetics of PYY₃₋₃₆ release from PEG₄₀-FMS-PYY₃₋₃₆.PEG₄₀-FMS-PYY₃₋₃₆ (750 μM in 0.1 M phosphate buffer pH 8.5, 2 mM NaN₃),was incubated at 37° C. Aliquots (100 μl) were withdrawn at theindicated times and free PYY₃₋₃₆ was measured by HPLC. The cumulativeamount of PYY₃₋₃₆ released is shown as a function of time. The amount ofPYY-36 in the initial conjugate was determined by acid hydrolysis of a20 μl aliquot, followed by amino acid analysis.

FIG. 20 shows the rate of PEG₄₀-FMS-PYY₃₋₃₆ hydrolysis in normal mouseserum. PEG₄₀-FMS-PYY₃₋₃₆ (0.5 μM) in normal mouse serum was incubated at37° C. Aliquots were withdrawn at the indicated times and the amount of2-PEG₄₀-9-sulfo-fulvene released from PEG₄₀-FMS-PYY₃₋₃₆ was determinedby HPLC and taken for calculating the rate of PEG₄O-FMS-PYY₃₋₃₆hydrolysis. The insert shows that PYY₃₋₃₆ degrades rapidly in normalmouse serum at 37° C. PYY₃₋₃₆ (50 nM) in normal mouse serum wasincubated at 37° C. At the indicated times, aliquots (0.1 ml) wereremoved, de-proteinated by 3 volumes of ethanol and the quantity ofPYY₃₋₃₆ was determined in the supernatants by HPLC.

FIG. 21 shows that PEG₄₀-FMS-PYY₃₋₃₆ elicits prolonged satiety. Theprotocol described in FIG. 15 was repeated, except that the micereceived SC either saline or PEG₄₀-FMS-PYY₃₋₃₆ (5 nmol/mouse) at theindicated times prior to re-feeding. Results are average of threeidentical experiments, normalized according to the saline control.

FIG. 22 shows the time course of reactivation of PEG₄₀₀₀₀-FMS-hGH uponincubation at pH 8.5, 37° C. PEG₄₀₀₀₀-FMS-hGH (1 mg/ml) was incubated in0.1 M phosphate buffer, 0.6% BSA and −2 mM NaN₃ at 37° C. Aliquots werewithdrawn at the indicated time points, and analyzed for their potenciesto displace ¹²⁵I-hGH from enriched hGH-receptor preparation extractedfrom rabbit liver plasma membranes. Native hGH displaces ¹²⁵I-hGH inthis assay half maximally at a concentration of 0.3±0.03 nM. An hGHderivative exhibiting half maximal displacement in this assay at aconcentration of 3.0±0.3 nM is considered to have 10% of the nativereceptor's binding potency. The insert shows the rate of release of hGHfrom PEG₄₀-FMS-hGH upon incubation at pH 8.5, 37° C. PEG₄₀₀₀-FMS-hGH (1mg protein) was incubated as described above. At the indicated timepoints, 0.1 ml aliquots withdrawn and subjected to analytical HPLCanalysis.

FIG. 23 shows the rate of hydrolysis of PEG-FMS conjugates, uponincubation at pH 8.5, 37° C. The concentration of PEG₄₀₀₀-FMS-exendin-4and PEG₄₀₀₀₀-FMS-hGH was determined for each time point by HPLC. Thelinear plot obtained indicates that the rate of hydrolysis is of firstorder reaction. The half-life time of the conjugates was calculated fromt_(1/2)=ln2/k, when k is the slope of the linear plot (h⁻¹).

FIG. 24 shows the pharmacokinetic profiles of subcutaneouslyadministered radiolabeled insulin and human serum albumin in rats. Att=0, groups of Wistar rats (170±5 g each, n=5 per group) were injectedsubcutaneously with 0.3 ml PBS (pH 7.4) and 0.5% BSA containing either10 μg 125I-insulin (specific activity 2800 cpm/ng) or 10 μg HSA(specific activity 1400 cpm/ng). Blood aliquots obtained from the tailvein at the indicated time points were spotted onto Whatman #3 paper andweighed immediately. Each paper was washed with 10% TCA and measured forits radioactive content. Results are expressed as ng of TCA precipitableprotein (insulin or HSA) per ml blood. Each point in the figurerepresents the arithmetic mean of 5 rats±SE. Arrows indicate the timesat which peak values were attained and the species t_(1/2) values.

FIGS. 25A-25B show the HPLC analysis of purified HSA-Fmoc-insulin beforeand after the release of insulin by hydrolysis. HPLC was conducted witha linear gradient from 0 to 100% of solution A (0.1% TFA) to solution B(acetinitrile-H2O, 75:25 in 0.1% TFA) over 10 minutes and then 4 minutesin solution B, using a Chromolith RP 18e (100×4 mm) column at a rate of3 ml/minutes. The effluent was monitored at 220 nm. (25A) PurifiedHSA-Fmoc-insulin (100 μg loaded); (25B) Purified HSA-Fmoc-insulinfollowing 4 hrs hydrolysis through incubation at pH 10.3, 25° C. Underthe same experimental conditions, insulin elutes with Rt=6.91 min andhas a surface area of 187,000±9,000 mav/μg insulin.

FIG. 26 shows the dosage-dependent stimulation of lipogenesis in ratadipocytes. Lipogenesis was carried out for 2 h at 37° C. in plasticvials containing 0.5 ml of fat cell suspension (1.5×105 cells) and 0.2mM [U-¹⁴C] glucose in the presence or absence of the indicatedconcentrations of native insulin or HSA-insulin conjugates. Results areexpressed as a percentage of maximal stimulation. Insulin (100 ng/ml)stimulated lipogenesis four to five-fold above basal levels.HSA-Fmoc-insulin (1 mg/ml) was taken as containing 24±3 μg insulin permg HSA in this assay (see Table 6). The ED₅₀ values for native insulin(0.3 ng/ml) for HSA-Fmoc-insulin (2.4 ng/ml) and for HSA-Benz-insulin(130 ng/ml) are indicated with arrows on the figure.

FIGS. 27A-27B show the rate of insulin release from HSA-Fmoc-insulin andreactivation of the conjugate upon incubation at pH 8.5, 37° C. Asolution of HSA-Fmoc-insulin (1 mg/ml) was incubated in 0.1 M phosphatebuffer at pH 8.5, 37° C. At the indicated time points, aliquots (100 μl)were analyzed by HPLC for the amount of released insulin (27A) and forbiological potency in rat adipocytes (27B). Results are expressed as theamount of insulin released per mg HSA-Fmoc-insulin. An aliquot ofHSA-Fmoc-insulin exhibiting ED₅₀ value=3.0 ng/ml in a lipogenic assaywas considered to have 10% the native biological potency.

FIGS. 28A-28B show the circulating glucose levels in mice following asingle subcutaneous or intraperitoneal administration ofHSA-Fmoc-insulin. Mice were injected intraperitoneally (28A) orsubcutaneously (28B) with Zn2+ free insulin (3 μg/mouse in 0.2 mlsaline) or HSA-Fmoc-insulin (0.4 mg/mouse). Blood glucose levels weredetermined at the indicated time points. Food was removed during theexperiment. Each point is the arithmetic mean of n=5 mice±SE.

FIG. 29 shows the effect of a single subcutaneous administration ofHSA-Fmoc-insulin on blood glucose levels in STZ-rats. STZ-rats receivedsaline solution (control group), Zn2+-free insulin (20 μg/STZ rat) orHSA-Fmoc-insulin (7 mg/STZ-rat). Blood glucose levels were determined atthe time points indicated in the Figure. Each point represents thearithmetic mean of the blood glucose levels of n=5 rats±SE.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new conceptual approach for delivery ofdrugs, particularly peptides and proteins of low or medium molecularweight, by natural or synthetic carriers, whereby the carrier moiety andthe drug residue are not linked directly to each other or the drugmolecule is not encapsulated within the carrier, as in standard drugdelivery using such carriers, but rather both residues are linked todifferent positions of a scaffold structure that is highly sensitive tobases and is removable under physiological conditions.

The carrier used in the present invention may be a protein such asalbumin or a modified albumin, e.g., cationized bovine serum albumin(CBSA) or cationized human serum albumin (CHSA), or a protein containingglobin-like domains having long half-life in circulation, e.g., ahemoglobin-like protein such as hemoglobin A or S.

In one embodiment, the protein carrier is albumin, namely, human serumalbumin (HSA). In another embodiment, the protein carrier is cationizedalbumin. Cationized albumin (pI greater than 8), unlike native albumin(pI approximately 4), enters cerebrospinal fluid (CSF) rapidly fromblood. This suggests that a specific uptake mechanism for cationizedalbumin may exist at the brain capillary wall, i.e. the blood-brainbarrier, and thus it may be used for brain targeting. Cationizedalbumins may be obtained, for example, by substituting anionic carboxylgroups with cationic aminoethyl-amide groups. The cationized albumin maybe linked to the drug through the scaffold containing the structure thatis highly sensitive to bases and is removable under physiologicalconditions or it may be conjugated with a polymer such aspoly(ethyleneglycol)-poly(lactide) (PEG-PLA) nanoparticles (CBSA-NP),designed for brain drug delivery. The CBSA is covalently conjugated withthe maleimide function group at the distal of PEG surrounding thenanoparticles. The cationized albumin may also be coupled to stericallystabilized liposomes.

In another embodiment, the carrier is a polymer carrier moiety such as,but not limited to, linear or branched polyethylene glycol (PEG) andblock copolymers thereof, poly(lactic acid) and copolymers thereof,polyesters having suitable functional groups based on polylactide (PLA),polyglycolide (PGA), polycaprolactone (PCL), and their copolymers, andpolyamides based on polymethacrylamide and their copolymers. All thesepolymers should have suitable functional groups for linking to ascaffold structure of the formula (i)-(iv) that is highly sensitive tobases and is removable under physiological conditions, preferablythrough a spacer. Examples of the polymer carriers include PEG,poly(lactic acid)-block-polyethylene glycol,N-(2-hydroxypropyl)methacrylamide (HMPA) copolymer with suitablefunctional groups or poly-D,L-lactide-co-glycolide (PLGA) nanoparticles.The functional groups may be hydroxy, amino, carboxyl, mercapto,sulfonic acid group, and the like.

The polymer may also be a block polymer as disclosed in U.S. Pat. No.5,929,177, herein incorporated by reference in its entirety as if fullydisclosed herein. These block polymers have functional groups, e.g.,amino group, carboxyl group or mercapto group on α-terminal, andhydroxyl group, carboxyl group, aldehyde group or vinyl group onω-terminal, and comprise hydrophilic/hydrophobic segments. Hydrophilicsegment comprises polyethylene oxide, while hydrophobic segment isderived from lactide, lactone or (meth)acrylic acid ester. These blockpolymers form polymeric micelles which are usable as bio-compatiblematerials.

The invention also encompasses as polymer carriers liposomes containingphospholipids with covalently attached poly(ethylene glycol)(PEG-lipids).

Polymer carriers have several advantages over other delivery methodssuch as liposomes and antibodies. Because liposomes—spherical vesiclesmade of phospholipids—are particles, they get taken up by macrophages.High levels can be found in the liver and spleen, even when theliposomes are given “stealth” characteristics by coating them with PEG.In contrast, water-soluble polymers allow working with a single moleculerather than a large particle. It is possible to choose a material whichdoesn't go to the liver and the spleen. It is in effect a‘macromolecular prodrug’. To avoid the liver and spleen, one can useuncharged hydrophilic polymers, such as PEG andN-(2-hydroxypropyl)-methacrylamide. When these polymers are hydrated,they can circulate in the blood for periods of up to about 24 hours.

In one preferred embodiment, the polymeric carrier is in the form ofnanoparticles. Nanoparticle drug delivery, utilizing degradable andabsorbable polymers, provides a more efficient, less risk solution tomany drug delivery challenges. Nanoparticles are generally defined asparticles between 10 nanometers (nm) and 1000 nm in size, and can beeither spherical or vesicular,

The advantages of using polymeric nanoparticles (PNPs) in drug deliveryare many, the most important being that they generally increase thestability of any volatile pharmaceutical agents and that they are easilyand cheaply fabricated in large quantities by a multitude of methods.Additionally, the use of absorbable or degradable polymers, such aspolyesters, provides a high degree of biocompatibility for PNP deliverysystems. Among the adaptations that can be made are surfacemodifications of the polymer, use of different fabrication methods,selection of a variety of pre-existing polymers or copolymers. In oneembodiment, the polymer carrier are nanoparticles of organicallymodified silica.

In one preferred embodiment of the invention, the polymer carrier isPEG. The present invention thus provides a new conceptual approach forreversible pegylation of drugs, particularly peptides and proteins oflow or medium molecular weight, whereby the PEG moiety and the drugresidue are not linked directly to each other, as in standardpegyylation procedures, but rather both residues are linked to differentpositions of a scaffold structure that is highly sensitive to bases andis removable under physiological conditions.

In one aspect, the present invention provides a compound of the formula:

(X)_(n)—Y

wherein

Y is a moiety of a drug bearing at least one functional group selectedfrom free amino, carboxyl, phosphate, hydroxyl and/or mercapto, and

X is a radical selected from the group of radicals consisting of theformulas (i) to (iv):

wherein:

R₁ is a radical containing a protein or polymer carrier moiety;polyethylene glycol (PEG) moiety;

R₂ is selected from the group consisting of hydrogen, alkyl, alkoxy,alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, —SO₃H, —SO₂NHR,amino, ammonium, carboxyl, PO₃H₂, and OPO₃H₂;

R is selected from the group consisting of hydrogen, alkyl and aryl;

R₃ and R₄, the same or different, are each selected from the groupconsisting of hydrogen, alkyl and aryl;

A is a covalent bond when the radical is linked to a carboxyl, phosphateor mercapto group of the drug Y, or A is OCO— when the radical is linkedto an amino or hydroxyl group of the drug Y;

n is an integer of at least one,

and pharmaceutically acceptable salts thereof.

The terms “alkyl”, “alkoxy”, “alkoxyalkyl”, “aryl”, “alkaryl” and“aralkyl” in the definitions of R₁, R₂, R₃ and R₄ herein are used todenote alkyl radicals of 1-8, preferably 1-4 carbon atoms, e.g. methyl,ethyl, propyl, isopropyl and butyl, and aryl radicals of 6-10 carbonatoms, e.g. phenyl and naphthyl. The term “halogen” includes bromo,fluoro, chloro and iodo.

In one preferred embodiment of the invention, X is a radical of theformula (i), more preferably a radical of formula (i) wherein R₂, R₃ andR₄ are each hydrogen and A is —OCO—, namely the9-fluorenylmethoxycarbonyl radical (hereinafter “Fmoc”), or mostpreferably, a radical of formula (i) wherein R₂ is —SO₃H at position 2of the fluorene ring, R₃ and R₄ are each hydrogen, and A is —OCO—,namely the 2-sulfo-9-fluorenylmethoxycarbonyl radical (hereinafter“FMS”).

In another embodiment of the invention, the functional group is theradical (i), wherein R₂, R₃ and R₄ are hydrogen and A is a covalentbond, i.e. the 9-fluorenylmethyl (Fm) group, which is applicable forreversible masking of free mercapto groups, of carboxylic functions ofaspartic and glutamic acid moieties, and of C-terminal carboxylfunctions of the cytokine molecules. The resulting 9-fluorenylmethylesters (Fm-esters) generate the parent free carboxylic functionsfollowing a β-elimination reaction pathway upon mild basic treatment,and thus can be similarly employed for reversible masking of carboxylicfunctions of drugs. The Fmoc-group is of further potential similar usein the reversible protection of hydroxyl groups of tyrosine, serine andthreonine.

The halogenated Fmoc radicals (i) wherein R₂ is halogen in the 2 or 7position, preferably Cl or Br, the 2-chloro-1-indenylmethoxycarbonyl(CLIMOC) radical (ii), the 1-benzo[f]indenylmethoxycarbonyl urethane(BIMOC) radical (iii), the urethane sulfone radical (iv) andcorresponding radicals (i) to (iv) wherein A is a covalent bond, can beused similarly to Fmoc and Fm for substitution of free amino, carboxyl,hydroxyl and mercapto functions of drugs, thus providing a wide range ofsensitivity toward removal of such groups under basic, e.g.physiological, conditions. In fact, the above radicals (i) to (iv)belong to a general family of rare chemical entities that undergohydrolysis at neutral or slightly alkaline pH and mild conditions, andcan therefore be used for temporary reversible protection of α- andε-amino groups, for example in peptide synthesis, and can be removedfrom the amino function by a β-elimination reaction, under mild basicconditions.

According to the invention, a radical (i) to (iv), preferably Fmoc orFMS covalently linked to amino and/or hydroxyl moieties or Fm covalentlylinked to carboxyl and/or mercapto moieties, undergoes hydrolysis (viaβ-elimination) back to the free amino, hydroxy, mercapto or carboxylfunctions, under physiological conditions in the body fluid, namely atpH 7.4 and 37° C.

In one embodiment, R₁ contains a protein carrier, preferably albumin,linked through a spacer to the ring. In another embodiment, R₁ containsa polymer carrier moiety; linked through a spacer to the ring.Preferably, the polymer carrier is a polyethylene glycol (PEG) moiety.

In one embodiment of the invention, R₁ is a radical of the formula:

—R₅—R₆-PEG

wherein

R₅ is selected from the group consisting of —NH—, —S—, —CO—, —COO—,—CH₂—, —SO₂—, —SO₃—, —PO₂—, and —PO₃—; and

R₆ is a bond or a radical by which the PEG moiety is covalently attachedto R₅.

In a more preferred embodiment, R₅ is —NH—, and R₆ is selected from thegroup consisting of —CO—, —COO—, —CH₂—, —CH(CH₃)—, CO—NH—, —CS—NH,—CO—CH₂—NH—CO—, —CO—CH(CH₃)—NH—CO—, —CO—CH₂—NH—CO—NH,

Z is O, S or NH; and

R₇ is selected from the group consisting of C1-C18 straight or branchedalkylene, phenylene, an oxyalkylene radical having 3-18 carbon atoms inthe backbone, a residue of a peptide containing 2-10 amino acidresidues, and a residue of a saccharide containing 1-10 monosaccharideresidues.

In the 4-chloro-6-Z-triazin-2-yl radical above, the 6-Z— group is linkedto the PEG moiety while the 2 position is linked to R₅, which is —NH— inthis case. In the —CO—R₇-succinimido radical above, the thio —S— groupat position 3 is linked to the PEG moiety while the —CO— is linked toR₅, which is —NH— in this case.

In one preferred embodiment, the pegylated drug compound of theinvention is a conjugate of the formula:

wherein R₂ is H or —SO₃H at position 2 of the fluorene ring, and Y ispreferably a peptide or protein drug. When R₂ is H, a herein designatedPEG-Fmoc-drug Y conjugate is obtained. In a most preferred embodiment,R₂ is —SO₃H at position 2 of the fluorene ring, and a herein designatedPEG-FMS-drug Y conjugate is obtained.

In a more preferred embodiment, the pegylated drug of the invention is acompound of the formula:

wherein R₂ is H or —SO₃H.

In a most preferred embodiment, the pegylated drug of the invention is acompound of the formula above, wherein R₂ is —SO₃H at position 2 of thefluorene ring, and the PEG moiety is a 40 kDa branched PEG. Theseconjugates are herein identified as (PEG₄O-FMS)_(n)-peptide/protein,wherein n is 1 to 3, preferably 1 or 2, most preferably 1.

The above compounds of the invention wherein R₅ is —NH— can be preparedfrom N-(9-fluorenylmethoxy-carbonyloxy)-succinimide (Fmoc-OSu) orN-(2-sulfo-9-fluorenylmethoxy-carbonyloxy)-succinimide (FMS-OSu)substituted by —NH₂ in the fluorene ring (depicted in Scheme 7, page a,first row, first column), by reacting the amino group with the activatedPEG-OH (e.g PEG-O—CO—Cl) or an activated derivatized PEG such asPEG-carboxylate (PEG-COOH, e.g. via PEG-CO—Cl), PEG-aldehyde (PEG-CHO),PEG-isocyanate (PEG-N═C═O), PEG-isothiocyanate (PEG-N═C═S),2,4-dichloro-6-S-PEG-1,3,5-triazine,2,4-dichloro-6-NH-PEG-1,3,5-triazine, or2,4-dichloro-6-O-PEG-1,3,5-triazine (Scheme 7, page a, right column) inorder to obtain the derivatives wherein —R₆—PEG is as presented inScheme 7 (page a, middle column).

In one preferred embodiment of the invention, the conjugate of theinvention is a (PEG-Fmoc)_(n)-peptide/protein, and the starting compoundfor their preparation is the maleimido derivative of Fmoc-OSu, hereindesignated Precursor 7 or MAL-Fmoc-NHS or MAL-Fmoc-OSu, of the formuladepicted in Scheme 3.

In a most preferred embodiment of the invention, the conjugate of theinvention is a (PEG-FMS)_(n)-peptide/protein, and the starting compoundfor their preparation is the maleimido derivative of FMS-OSu, hereindesignated Precursor 8 or MAL-FMS-NHS or MAL-FMS-OSu, of the formuladepicted in Scheme 3.

Two possible pathways for the pegylation of target peptides/proteins andpreparation of the (PEG-Fmoc)_(n)-peptide/protein or(PEG-FMS)_(n)-peptide/protein conjugates are provided by the invention,as depicted in Scheme 6. Both pathways are two-step procedures.

According to one pathway, MAL-FMS-NHS or MAL-Fmoc-NHS is first attachedto the amine component of the target peptide/protein, thus obtaining aMAL-FMS-peptide/protein or MAL-Fmoc-peptide/protein conjugate, and thensubstituting PEG-SH for the maleimide moiety, producing the(PEG-FMS)_(n)-peptide/protein or (PEG-Fmoc)_(n)-peptide/proteinconjugate, respectively.

In the second pathway, MAL-FMS-NHS or MAL-Fmoc-NHS is first reacted withPEG-SH, thus forming a PEG-FMS-NHS or PEG-Fmoc-NHS conjugate, and thenreacting it with the amine component of the target peptide or proteinresulting in the desired (PEG-FMS)_(n)-peptide/protein or(PEG-Fmoc)_(n)-peptide/protein conjugate, respectively. This pathway issuitable for sulfhydryl- or disulfide-containing peptides and proteins.

The compounds wherein R₅ is —NH— and R₆ is —CO—NH— or —CS—NH— can beprepared from Fmoc-OSu or FMS-OSu substituted by —N═C═O or —N═C═S in thefluorene ring (depicted in Scheme 7, page C, first row and last rows,first column), respectively, by reaction with PEG-NH₂. No activation isnecessary because these Fmoc/FMS species are already activated.

In a further embodiment of the invention, R₅ is —S—; and R₆ is selectedfrom the group consisting of

wherein Z is O, S or NH.

The above compounds of the invention wherein R₅ is —S— can be preparedfrom Fmoc-OSu or FMS-OSu substituted by —SH in the fluorene ring(depicted in Scheme 7, page b, second row, first column), by reactionwith PEG-maleimide of the formula shown in Scheme 7, page b, second row,right column), thus obtaining a pegylated compound of the inventionwherein the PEG moiety is linked to the fluorene ring trough a residueas depicted in Scheme 7, page b, second row, middle column, or byreaction with 2,4-dichloro-6-S-PEG-1,3,5-triazine,2,4-dichloro-6-NH-PEG-1,3,5-triazine, or2,4-dichloro-6-O-PEG-1,3,5-triazine.

In yet another embodiment, in the pegylated drug of the invention, R₅ is—CO; R₆ is selected from the group consisting of —O—; —NH—; —NH—R₇—COO—;—NH—R₇ —NH; —NH—R₇—CO—NH

Z is O, S or NH; and

R₇ is selected from the group consisting of C1-C18 straight or branchedalkylene, phenylene, an oxyalkylene radical having 3-18 carbon atoms inthe backbone, a residue of a peptide containing 2-10 amino acidresidues, and a residue of a saccharide containing 1-10 monosaccharideresidues.

The above compounds of the invention wherein R₅ is —CO— can be preparedfrom Fmoc-OSu or FMS-OSu substituted by —COOH in the fluorene ring(depicted in Scheme 7, page b, third row, first column). When R₆ is —O—or —NH—, the reaction will occur with PEG (PEG-OH) or PEG-amine(PEG-NH₂), respectively, thus obtaining a pegylated compound wherein thePEG moiety is linked to the fluorene ring trough a residue —CO—O— or—CO—NH—, respectively, as depicted in Scheme 7, page b, middle column(3^(rd) and 4^(th) rows).

The compounds of the invention wherein R₅ is —CO— and R₆ is NH—R₇—COO—can be prepared from Fmoc-OSu or FMS-OSu substituted by —COOH in thefluorene ring, by reaction with H₂N—R₇—CO-OtBu using a coupling reagent(e.g. DCC/HOBt, or PyBOP(benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexa-fluorophosphate)/triethylamine), removal of the tBu protectinggroup under acidic conditions (e.g. trifluoroacetic acid or HCl indioxane), activation of the free carboxyl group by triphosgen andreaction of the —NH—R₇—COCl formed with PEG-OH to obtain the—CO—NH—R₇—CO—O-PEG derivative.

The compounds of the invention wherein R₅ is —CO— and R₆ is NH—R₇—NH canbe prepared from Fmoc-OSu or FMS-OSu substituted by —COOH in thefluorene ring, by reaction with H₂N—R₇—NH-tBu using a coupling reagentand removing the tBu protecting group as described above, reacting thefree amino group with PEG-OSu to obtain the —CO—NH—R₇—NH-PEG derivative.

The compounds of the invention wherein R₅ is —CO— and R₆ is NH—R₇—CO—NH—can be prepared from Fmoc-OSu or FMS-OSu substituted by —COOH in thefluorene ring, by reaction with H₂N—R₇—CO—NH-tBu using a couplingreagent and removing the tBu protecting group as described above,activating the free carboxyl group with DCC/NHS and reacting of the—NH—R₇—CO—N-hydroxysuccinimide ester formed with PEG-NH₂ to obtain the—CO—NH—R₇—CO—NH-PEG derivative.

The compounds of the invention wherein R₅ is —CO— and R₆ is—NH—R₇—NH—Z-(4-chloro-6-Z-PEG-1,3,5-triazin-2-yl can be prepared fromFmoc-OSu or FMS-OSu substituted by —COOH in the fluorene ring, byreaction with H₂N—R₇—NH-tBoc or H₂N—R₇—O-tBu or H₂N—R₇—S-trityl using acoupling reagent and removing the tBoc, tBu or trityl protecting groupas described above, reacting the free NH₂, OH or SH group with2,4,6-trichloro-1,3,5-triazine, and further reacting the —NH—R₇—NH[O orS]-(4-chloro-6-NH[O or S]-1,3,5-triazin-2-yl thus formed with PEG-NH₂,PEG-OH or PEG-SH to obtain the corresponding —CO—NH—R₇—NH[O orS]-(4-chloro-6-NH[O or S]-PEG-1,3,5-triazine derivative.

The compounds of the invention wherein R₅ is —CO— and R₆ isNH—R₇-ethylene-succinimido can be prepared from Fmoc-OSu or FMS-OSusubstituted by —COOH in the fluorene ring, by reaction withH₂N—R₇-ethylene-maleimide using a coupling reagent (e.g. DCC/HOBt,PyBoP/Triethylamine) followed by reaction of the maleimide moiety withPEG-SH at pH 6-8 to obtain the —CO—NH—R₇-ethylene succinimido-S-PEGderivative.

The pegylated-Fmoc/FMS-drugs of the invention are then prepared fromthese intermediates by the one-step procedure described in Example 5hereinafter.

In yet a further embodiment of the invention, R₅ is —CH₂—; and R₆ is—(CH₂)_(n)—S— or —(CH₂)_(n)—NH—, wherein n is 0 to 18, preferably 1.

The above compounds of the invention wherein R₅ is —CH₂— and R₆ is—CH₂—S— or —R₆ is —CH₂—NH— can be prepared from Fmoc-OSu or FMS-OSusubstituted by —COH in the fluorene ring (depicted in Scheme 7, page c,4^(th) row, first column), by reaction with PEG-SH or PEG-NH₂ followedby reduction with NaHBH₃, respectively.

The above compounds of the invention wherein R₅ is —CH₂— and 1% is—(CH₂)_(n)—S— or R₆ is —(CH₂)_(n)—NH— can be prepared from Fmoc-OSu orFMS-OSu substituted by —(CH₂)_(n)-Hal in the fluorene ring, wherein Halis F, Cl, Br or I (depicted in Scheme 7, page d, 3^(rd) row, firstcolumn), by reaction with PEG-NH₂ or PEG-SH, thus obtaining a pegylatedcompound of the invention wherein the PEG moiety is linked to thefluorene ring trough a residue as depicted in Scheme 7, page d, middlecolumn, 3^(rd) and 4^(th) rows, respectively.

The pegylated-Fmoc/FMS-drugs of the invention are then prepared from theabove intermediates by the one-step procedure described in Example 5hereinafter.

In still another embodiment, R₅ is —SO₂— and R₆ is —O—, —NH— or—CH₂—CH₂—S.

The compounds wherein R₆ is —O— or —NH— can be prepared from Fmoc-OSu orFMS-OSu substituted by —SO₂Cl in the fluorene ring (depicted in Scheme7, page c, first column, 3rd row), by reaction with PEG-OH or PEG-NH₂,respectively. The pegylated-Fmoc/FMS-drugs of the invention are thenprepared from these intermediates by the one-step procedure described inExample 5 hereinafter.

The compounds wherein R₆ is —CH₂—CH₂—S, can be prepared from Fmoc-OSu orFMS-OSu substituted by —SO₂CH═CH₂ in the fluorene ring (depicted inScheme 7, page c, first column, 2nd row), by reaction with PEG-SH. Thepegylated-Fmoc/FMS-drugs of the invention are then prepared from theseintermediates by the two-step procedure described in Examples 16 or 17hereinafter.

In yet still another embodiment, R₅ is —PO₂— and R₆ is —O— or —NH—.These compounds can be prepared from Fmoc-OSu or FMS-OSu substituted by—PO₂Cl in the fluorene ring (depicted in Scheme 7, page d, first column,first row), by reaction with PEG-OH or PEG-NH₂, respectively. Thepegylated-Fmoc/FMS-drugs of the invention are then prepared from theseintermediates by the one-step procedure described in Example 5hereinafter.

According to the present invention, Y is a moiety of a drug bearing atleast one functional group selected from free amino, carboxyl, hydroxyl,phosphate and/or mercapto. In a more preferred embodiment of theinvention, the drug contains at least one free amino group and is apeptide or a protein drug or a non-peptidic drug.

In one embodiment of the invention, the drug is a non-peptidic drug thatcontains at least one amino group and the invention relates to PEG-Fmocand PEG-FMS conjugates thereof. Non-peptidic drugs that are amenable tothe technology of the invention include antibiotic aminoglycosides suchas gentamicin and amphotericin B, and antineoplastic drugs such asaminolevulinic acid, daunorubicin and doxorubicin.

In a more preferred embodiment of the invention, the drug containing atleast one amino group is a peptide or protein drug, most preferably apeptide or protein of low or medium molecular weight, that can be usedas a drug for human or veterinary use.

Thus, further provided by the invention are pegylated drug conjugatesPEG-FMS-Y and PEG-Fmoc-Y herein identified by the formulas:

(PEG-FMS)_(n)-Y or (PEG-Fmoc)_(n)-Y

wherein Y is a moiety of a peptide or protein drug, n is an integer ofat least one, preferably 1 or 2, and Y is linked to the FMS or Fmocradical through at least one amino group. In the most preferredembodiment the conjugate is (PEG-FMS)_(n)—Y and the PEG moiety isPEG₄₀₀₀₀.

Examples of peptides and proteins Y that can be pegylated according tothe invention include, but are not limited to, insulin, an interferonsuch as IFN-α2, a PYY agonist such as the peptide PYY₃₋₃₆, an exendinsuch as exendin-3 and exendin-4 and exendin analogues and agonists,atrial natriuretic peptide (ANP), human growth hormone (hGH),erythropoietin, TNF-α, calcitonin, gonadotropin releasing hormone (GnRH)or an analogue thereof such as leuprolide and D-Lys⁶-GnRH, hirudin,glucagon, and a monoclonal antibody fragment such as anti-TNFαmonoclonal antibody fragment.

In one preferred embodiment of the invention, the peptidic drug isinsulin and the invention provides PEG-Fmoc-insulin and PEG-FMS-insulinconjugates and pharmaceutical compositions comprising them for treatmentof diabetes mellitus and hyperglycemia. Examples of such conjugates arethe (PEG₅₀₀₀-Fmoc)₁-insulin, (PEG₅₀₀₀-Fmoc)-2-insulin andPEG₄₀₀₀₀-FMS-insulin conjugates.

In another preferred embodiment of the invention, the drug is an exendinor an exendin agonist.

In a more preferred embodiment, the drug is exendin-4 of the sequencerepresented by SEQ. ID. NO: 1:

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH₂

In another preferred embodiment, the drug is exendin-3 of the sequencerepresented by SEQ. ID. NO:2:

HSDGTFITSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH₂

In a further preferred embodiment, the drug is an exendin agonistdefined herein as a compound that mimics the activities of exendin-3 orexendin-4 by binding to the receptor(s) at which exendin-3 or exendin-4exerts its actions which are beneficial as insulinotropic and in thetreatment of diabetes mellitus or by mimicking the effects of exendin onincreasing urine flow, increasing urinary sodium excretion and/ordecreasing urinary potassium concentration, by binding to thereceptor(s) where exendins cause these effects. Preferably, the exendinagonist is selected from the group of insulinotropic exendin-4 fragmentsand analogues consisting of exendin agonists represented by SEQ ID NO:3to SEQ ID NO: 10:

exendin-4 (1-31) [SEQ ID No: 3] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGP;Y³¹exendin-4(1-31) [SEQ ID No: 4] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGY;exendin-4 (1-30) [SEQ ID No: 5] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG;exendin-4 (1-30) amide [SEQ ID No: 6]HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG-NH₂; exendin-4 (1-28) amide [SEQ ID No:7] HGEGTFTSDLSKQMEEEAVRLFIEWLKN-NH₂; L¹⁴, F²⁵exendin-4 amide [SEQ ID No:8] HGEGTFTSDLSKQLEEEAVRLFIEFLKNGGPSSGAPPPS-NH₂; L¹⁴, F²⁵ exendin-4(1-28) amide [SEQ ID No: 9] HGEGTFTSDLSKQLEEEAVRLFIEFLKN-NH₂; and L¹⁴,A²², F²⁵ exendin-4 (1-28) amide [SEQ ID No: 10]HGEGTETSDLSKQLEEEAVRLAIEFLKN-NH₂.

According to this embodiment, the invention providesPEG-Fmoc-exendin/exendin agonist and PEG-FMS-exendin/exendin agonistconjugates and pharmaceutical compositions comprising them forprevention of hyperglycemia and for treatment of diabetes mellitusselected from the group consisting of non-insulin dependent diabetesmellitus, insulin-dependent diabetes mellitus, and gestational diabetesmellitus. In a most preferred embodiment, the pegylated exendinconjugate of the invention is PEG₄₀-FMS-exendin-4.

In a farther preferred embodiment of the invention, the peptidic drug isan interferon, preferably IFN-α, and the invention providesPEG-Fmoc-IFN-α and PEG-FMS-IFN-α conjugates and pharmaceuticalcompositions comprising them for treatment of diseases treatable byIFN-α, particularly viral diseases, more particularly hepatitis B or C,both as sole therapy or in conjunction with an antiviral agent such asribavirin, or for treatment of cancer, e.g. transitional cell carcinoma,the most common type of bladder cancer, ovarian cancer, pancreaticcancer melanoma, non-Hodgkin's lymphoma, hairy cell leukemia, andAIDS-related Kaposi's sarcoma, both as sole therapy or in conjunctionwith a cytotoxic agent such as carboplatin and/or cyclophosphamide. In amore preferred embodiment, the conjugate is (PEG₄₀-FMS)₂—IFNα2 or, mostpreferably, PEG₄₀-FMS-IFNα2.

In still another preferred embodiment of the invention, the peptidicdrug is a PYY agonist, herein defined as a molecule that has a PYY- orPYY[3-36]-like biological activity such as reducing food intake inmammals, and acts by a mechanism similar to that of PYY and PYY[3-36],for example by binding to the Y2 receptor. The PYY agonist is preferablyan agonist specific for the Y2 receptor and is preferably a peptidecontaining, at a minimum, the sequence of amino acids 25-36 of PYY, mostpreferably the sequence 3-36 of PYY.

In one embodiment of the invention, the PYY agonist is the 36-merpeptide

PYY of the sequence represented by SEQ ID NO: 11:

YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH₂In a more preferred embodiment of the invention, the PYY agonist is thepeptide PYY[3-36] of the sequence represented by SEQ ID NO: 12:

IKPEAPGEDASPEELNRYYASLRHYLNLVTYRQRY-NH₂

According to this embodiment, the invention provides PEG-Fmoc-PYYagonist and PEG-FMS-PYY agonist conjugates and pharmaceuticalcompositions comprising them for particularly for reduction of foodintake, for inducing weight loss and for the treatment of diseases ordisorders which can be alleviated by reduction of food intake such asobesity, hypertension, dyslipidemia, cardiovascular risk,insulin-resistance, and diabetes mellitus (particularly type IIdiabetes). In a preferred embodiment, the pegylated PYY agonist of theinvention is PEG₄O-FMS-PYY₃₋₃₆.

In another preferred embodiment, the peptidic drug is human growthhormone (hGH) and the invention provides PEG-Fmoc-hGH and PEG-FMS-HGHconjugates and pharmaceutical compositions comprising them for treatmentof conditions and disorders treatable by hGH, particularly for treatmentof children of pathologically short stature and as anti-aging agent. Inpreferred embodiments, the pegylated hGH conjugates of the invention are(PEG₄₀-FMS)-2-hGH and PEG-FMS-hGH.

In a further preferred embodiment of the invention, the peptidic drug isatrial natriuretic peptide (ANP) or an analog thereof, particularly thecyclic 28-amino acid ANP of the sequence represented by SEQ ID NO:13, asfollows:

Ser-Leu-Arg-Arg-Ser-Ser-[Cys7-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys23]- Asn-Ser-Phe-Arg-Tyr

According to this embodiment, the invention provides PEG-Fmoc-ANP andPEG-FMS-ANP conjugates and pharmaceutical compositions comprising themfor treatment of conditions and disorders treatable by the inventionprovides PEG-Fmoc-ANP and PEG-FMS-ANP conjugates and pharmaceuticalcompositions comprising them for treatment of conditions and disorderstreatable by natriuretic peptides and variants thereof, particularlytreatment of cardiovascular diseases, congestive heart failure,hypertension, acute kidney failure and adult respiratory distresssyndrome (ARDS). In one preferred embodiment of the invention, thepegylated ANP is the PEG₄₀-FMS-ANP conjugate.

Also included in the scope of the invention are pharmaceuticallyacceptable salts of the pegylated conjugates of the invention. As usedherein, the term “salts” refers to both salts of carboxyl groups and toacid addition salts of amino groups of the drug, e.g. peptide orprotein, molecule. Salts of a carboxyl group may be formed by meansknown in the art and include inorganic salts, for example, sodium,calcium, ammonium, ferric or zinc salts, and the like, and salts withorganic bases such as those formed for example, with amines, such astriethanolamine, arginine, or lysine, piperidine, procaine, and thelike. Acid addition salts include, for example, salts with mineral acidssuch as, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid.

The present invention further relates to methods for the preparation ofthe pegylated conjugates of the invention and to several novel precursorcompounds used in these methods.

Thus, the present invention also relates to a precursor of the formula:

wherein:

R₁ is a radical of the formula —R₅—R₆—B;

R₂ is H or —SO₃H at position 2 of the fluorene ring;

B is maleimido, —S—CO—CH₃ or a PEG moiety;

R₅ is selected from the group consisting of —NH—, —S—, —CO—, —COO—,—CH₂—, —SO₂—, —SO₃—, —PO₂—, and —PO₃—; and

R₆ is a bond or a radical by which the maleimido, —S—CO—CH₃ or PEGmoiety is attached to R₅.

In one preferred embodiment, R₅ is NH—; R₆ is selected from the groupconsisting of —CO—, —COO—, —CH₂—, —CH(CH₃)—, CO—NH—, —CS—NH—,—CO—CH₂—NH—CO—, —CO—CH(CH₃)—NH—CO—, —CO—CH₂—NH—CO—NH, —CO—R₈—;

Z is O, S or NH;

R₇ is selected from the group consisting of C1-C18 straight or branchedalkylene, phenylene, an oxyalkylene radical having 3-18 carbon atoms inthe backbone, a residue of a peptide containing 2-10 amino acidresidues, and a residue of a saccharide containing 1-10 monosaccharideresidues; and

R₈ is a C1-C8 straight or branched alkylene, preferably ethylene, when Bis maleimido or —S—CO—CH₃.

In one preferred embodiment, the invention relates to the novelPrecursors 1-7, whose formulas are depicted in Schemes 1 and 3. In themost preferred embodiment, the invention relates to the compound hereinidentified as Precursor 8 or MAL-FMS-NHS of the formula:

Precursor 8 is the compoundN-[2-(maleimido-propionylamino)-7-sulfo-fluoren-9-yl-methoxycarbonyloxy]-succinimide[or9-hydroxymethyl-2-(amino-3-maleimidopropionate)-7-sulfo-fluorene-N-hydroxysuccinimide]and is also herein identified as MAL-FMS-OSu.

MAL-FMS-NHS is a water-soluble hetero-bifunctional reagent, consistingof a sulfonated fluorenyloxycarbonyl N-hydroxysuccinimide ester thatreacts with peptide and protein amino groups. A maleimide group wasattached to the fluorenyl backbone to enable coupling tosulfhydryl-containing PEG, most preferably PEG₄₀₀₀₀-SH.

Thus, further provided by the invention are precursor drug conjugatesherein identified by the formula:

(MAL-FMS)_(n)-Y or (MAL-Fmoc)_(n)-Y

wherein Y is a moiety of a drug, more preferably a peptide or proteindrug, n is an integer of at least one, preferably 1 or 2, and Y islinked to the FMS or Fmoc radical through an amino group.

The present invention also provides a method for the preparation of aconjugate (PEG-Fmoc)_(n)-Y, wherein Y is a moiety of a drug, morepreferably a peptide or protein drug, n is an integer of at least one,preferably 1 or 2, and Y is linked to the Fmoc radical through an aminogroup, which comprises:

(i) reacting a drug Y, e.g. a peptide or protein drug, with at least oneequivalent of Precursor 7, thus obtaining a conjugate (MAL-Fmoc)_(n)-Y;and

(ii) reacting the conjugate (MAL-Fmoc)_(n)-Y with PEG-SH.

In a most preferred embodiment, the invention relates to a method forpreparation of a conjugate (PEG-FMS)_(n)—Y, wherein Y is a moiety of adrug, more preferably a peptide or protein drug, n is an integer of atleast one, preferably 1 or 2, and Y is linked to the FMS radical throughan amino group, which comprises:

(i) reacting a drug Y, e.g. a peptide or protein drug, with at least oneequivalent of MAL-FMS-NHS, thus obtaining a conjugate (MAL-FMS)_(n)—Y;and

(ii) reacting the conjugate (MAL-FMS)_(n)—Y with PEG-SH, thus obtainingthe conjugate (PEG-FMs)_(n)-Y.

In another most preferred embodiment, the invention relates to a methodfor preparation of a conjugate (PEG-FMS)_(n)—Y, which comprises:

(i) reacting MAL-FMS-NHS with PEG-SH, thus obtaining a conjugatePEG-FMS-NHS; and

(ii) reacting the drug Y. e.g. a peptide or protein drug, with at leastone equivalent of the conjugate PEG-FMS-NHS, thus obtaining theconjugate (PEG-FMS)_(n)—Y.

The PEG-SH reagent is preferably PEG₄₀-SH, wherein the PEG moiety is abranched PEG moiety of molecular weight 40,000 Da.

As mentioned in the Background section herein, the pegylation techniquehas been extensively used for modifying molecules, in particular peptideand protein drugs, in an attempt to improve some of its characteristicssuch as improved stability and solubility, reduced immunogenicity,reduced proteolysis, reduced toxicity, reduced clearance by the kidneys,improved bioavailability, and extended circulating life thus lessfrequent dosing being required. However, one of the main problems ofpegylation is that covalent bonding between the PEG moiety and the drugmost often causes loss of biological activity or drastic decrease ofpharmacological potency of the drug. For this reason, pegylation is usedmore for high-molecular weight proteins, that are less likely to beinactivated by the reaction with PEG, but is less frequent for peptidesand low-molecular weight proteins.

Theoretically, decreased bioactivity of peptides and proteins bypegylation can be overcome by linking the PEG-chains via a chemical bondsensitive to mild alkaline and/or acid hydrolysis, or enzymaticallycleavable by serum proteases or esterases. Obviously, an inconsistentrate of hydrolysis would render such an approach impractical. Aprerequisite condition is therefore that the hydrolysis of thePEG-chains from the conjugate take place at a slow rate and in ahomogenous fashion under the strictly homeostatic pH and temperatureconditions of the mammalian circulatory system.

Previously, we have prepared 2-sulfo-9-fluorenylmethoxycarbonyl-Nhydroxysuccinimide (FMS-OSu) (Gershonov et al., 1999, 2000; Shechter etal., 2001, 2001a) as a reversible protein modifier. Protein-linked FMSmoieties undergo slow and spontaneous hydrolysis under physiologicalconditions generating the unmodified parent molecule. Hydrolysis ofFMS-protein conjugates at 37° C. in normal human serum, in aqueousbuffers of pH 8.5, or in the circulatory system in vivo, takes placewith a t_(1/2) of 5-7 hrs. Using the FMS moiety as the scaffold for thesuggested reversible pegylation technology enables the hydrolysis ratesof various PEG-FMS-protein conjugates to be predicted. The t_(1/2) forthe hydrolysis of modified small molecules and PEG-conjugatedpolypeptides and proteins falls within a relatively narrow range of 8-14h. The constant hydrolysis rate for PEG-FMS-protein conjugates is due tothe β-elimination reaction, which occurs at position 9 of the fluorenylmoiety, being solely dependent on the pH of the surrounding medium.Thus, in contrast to what occurs in approaches based on enzymatichydrolytic bond cleavage, similar PEG-FMS hydrolysis rates are expectedfrom all conjugates, regardless of the identity of the protein/peptidemoiety conjugated. Using the present approach one can further controlthe hydrolysis rate by substitution of the fluorenyl moiety withelectron-withdrawing or electron-inducing groups that increase ordecrease, respectively, the hydrolysis rate. The number of PEG-FMSchains attached to the drug should also affect the rate at which thenative drug is released.

The sulfonated fluorene moiety is not toxic, as previously shown(Shechter et al, 2001). High molecular-weight PEG is known to be safe interms of toxicity and immunogenicity and is widely used in the food,cosmetic and pharmaceutical industries (Working et al., 1997; Roberts etal., 2002).

Thus, the present invention provides a procedure herein designated“reversible pegylation”. In this new conceptual approach, that wasimplemented according to the invention with low molecular-weightpolypeptides and proteins, the PEG moiety is not attached directly tothe drug, as in the standard pegylation procedure, but rather the PEGmoiety is attached directly or through a linker to a moiety of formula(i) to (iv) herein, and the drug is attached to another position of themoiety (i) to (iv). Said moiety (i) to (iv), preferably the Fmoc or FMSmoiety (i), is highly sensitive to bases and is removable under mildbasic conditions. Thus, in this way, a prodrug is obtained that isinactive, but undergoes transformation into the active drug under thephysiological conditions of the body. The prodrug has an extendedcirculation life but the PEG moiety is removed together with the Fmoc orFMS moiety and the drug recovers its full pharmacological potency.

This novel approach enables the desirable pharmacological featuresassociated with pegylation to be conferred on low molecular-weightpeptide and protein drugs that would otherwise have been fully orpartially inactivated by this technique. A pharmacologically ‘silent’conjugate that is ‘trapped’ in the circulatory system releases thecovalently-linked parent peptide or protein, with a desirablepharmacokinetic profile. This new approach is expected to extend thelife-time, bioavailability and efficacy of existing peptide drugs, andto extend the same in known peptide drugs and peptide drug candidatesthat may yet be discovered.

This new technology has been successfully tested according to theinvention on several peptide and protein drugs. In addition toprolonging life-times in vivo, the inactive but reactivatablePEG-protein conjugate has the profound advantage of maintaining a lowcirculating level of the active protein drug at any time point afteradministration. In this way, a well-known risk of the presence of a“burst” of toxifying or desensitizing drug in the circulation isavoided.

As mentioned above, in theory one can design PEG chains that can bereleased from PEG-protein conjugates by serum proteases or esterases.However, rapid or unpredictable rate of release is not useful. Aprerequisite condition with this new technology is that the PEG-chainsshould be hydrolyzed spontaneously from the conjugates at a slow,continuous and predictable rate. Release of PEG-chains should occur overa prolonged period, thus maintaining the projected conjugates in thecirculatory system, prior to removal of PEG-chains by hydrolysis. Thepresent invention fulfills these requirements. For example, uponincubation in normal human serum at 37° C., PEG-chains are hydrolyzedfrom proteins, with a half-time of 8.0±2 hrs. Rates of release aredictated exclusively by the nature of the Fmoc-moiety (Fmoc/FMS), by thepH and the reactivity of the blood serum, and mammals maintain stricthomeostasis with regards to these last two parameters.

According to the present invention, a pilot experiment includedsynthesis of a pegylated insulin. Upon incubation of(PEG₅₀₀₀-Fmoc)₁-insulin derivative at pH 8.5, or at normal human serumat 37° C., the lipogenic activity was restored with a half-life of 30±2hrs. Regeneration of the lipogenic potency of bis-modified insulin,(PEG₅₀₀₀-Fmoc)-2-insulin, followed an additional lag period of 10±1 hrs.

A single subcutaneous administration of (PEG₅₀₀₀-Fmoc)₁-insulin in mice,lowered circulating glucose levels and the half time of return tonormoglycemic values exceeded 6.7 fold that for native insulin.Following intraperitoneal administration of (PEG₅₀₀₀-Fmoc)₁-insulin, thereturn to normoglycemia was 3.4 fold slower than after administration ofthe native hormone. In sum, a prototype of a reversible PEG has beenestablished. It undergoes slow spontaneous hydrolysis after conjugation,regenerating the non-modified parent drugs, at physiological conditions.Thus, the principal drawback of inactivating drugs by pegylation issolved by the technology of the present invention. The PEG-FMS moietieshydrolyze at faster rates as compared to PEG-Fmoc-moieties (t_(1/2)=5-7hrs, ref.). Compounds containing FMS-PEG moieties are more suitable when2-5 PEG-chains are to be introduced into a peptide or a protein drug,for obtaining the desirable pharmacological features of the conjugates.

In another example of the present invention, it is shown herein thatwhen native exendin-4 was subcutaneously administered at a dose of 4μg/mouse, the blood glucose level (BGL) declined by 26-28% (from 140mg/dl to 104-101 mg/dl), with the largest percent change in BGLoccurring 0.5-1 h after administration. Glucose concentrations thenreturned to their initial levels with a t_(1/2) of 3.7±0.3 h. Followingthe subcutaneous administration of PEG₄₀₀₀₀-FMS-exendin-4, the decreasein BGL took place at a more moderate rate. Circulating glucose reachedits lowest concentration 8-12 hours after administration (92 mg/dl,33%). Stable, low circulating glucose concentrations were thenmaintained for a further 12 hours. Return to initial glucose levels tookplace with a t_(1/2) of 30±2 h, being 7.5 times longer than thatobtained by the same dose of the native exendin-4.

In a further example of the present invention, it is shown herein thatusing a BIAcore binding assay, the in vitro rate of regeneration ofnative interferon was estimated to have a half-life of 65 hrs. Followingsubcutaneous administration to rats and monitoring circulating antiviralpotency, active IFNα2 levels peaked at 50 hrs, with substantial levelsstill being detected 200 hrs post administration. This value contrastswith a half-life of about 1 hr measured for unmodified interferon. Theconcentration of active IFNα2 scaled linearly with the quantityinjected. Comparing subcutaneous to intravenous administration ofPEG₄₀-FMS-IFNα2, we found that the long circulatory lifetime of IFNα2was affected both by the slow rate of absorption of the pegylatedprotein from the subcutaneous volume and by the slow rate of dischargefrom the PEG in circulation. A numerical simulation of the results wasin good agreement with the results observed in vivo. The pharmacokineticprofile of this novel pegylated IFNα2 conjugate combines a prolongedmaintenance in vivo with the regeneration of active-native IFNα2,ensuring ready access to peripheral tissues and thus an overalladvantage over currently used formulations.

Peptide YY₃₋₃₆ (PYY₃₋₃₆) was recently shown to induce satiety in miceand humans. It is described herein that the satiety induced bysubcutaneous administration of PYY₃₋₃₆ to fasting mice had a half-lifeof ˜3 h. Pegylation of PYY₃₋₃₆ through a non-hydrolysable bond yieldedan inactive conjugate but the conjugate of the inventionPEG₄₀-FMS-PYY₃₋₃₆ gradually released unmodified PYY₃₋₃₆ underphysiological conditions. Subcutaneous administration ofPEG₄₀-FMS-PYY₃₋₃₆ to mice resulted in protracted satiety, with ahalf-life of ˜24 h. PEG₄₀-FMS-PYY₃₋₃₆ can therefore serve as along-acting prodrug of PYY₃₋₃₆, thereby providing a more practical meansfor controlling human obesity.

The PEG moiety according to the invention may be linear or branched PEGand has a molecular weight in the range of 200 to 200,000 Da, preferablyup to 80,000 Da, more preferably 5,000-40,000 Da, and most preferablybetween about 20,000 Da and 40,000 Da. Preferably, the PEG moiety is abranched PEG molecule of 40,000 Da.

The prodrugs of the present invention are prepared using PEGylatingagents, meaning any PEG derivative which is capable of reacting with afunctional group such as, but not limited to, NH₂, OH, SH, COOH, CHO,—N═C═O, —N═C═S, —SO₂Cl, —SO₂CH═CH₂, —PO₂Cl, —(CH₂)_(x) Hal, present atthe fluorene ring of the Fmoc or FMS moiety. Examples of these reagentsand of the products obtained are depicted in Scheme 7 herein. Thesederivatized PEGs that can be used according to the invention and similarreagents are commercially available. It should be noted that thePEGylating agent is usually used in its mono-methoxylated form whereonly one hydroxyl group at one terminus of the PEG molecule is availablefor conjugation. However, a bifunctional form of PEG where both terminiare available for conjugation may be used if, for example, it is desiredto obtain a conjugate with two peptide or protein residues covalentlyattached to a single PEG moiety.

In a preferred embodiment of the invention, the PEG moiety is branched.Branched PEGs are in common use. They can be represented asR(PEG-OH)_(m) in which R represents a central core moiety such aspentaerythritol or glycerol, and m represents the number of branchingarms. The number of branching arms (m) can range from three to a hundredor more. The hydroxyl groups are subject to chemical modification.

The use of branched PEG molecules has several advantages including thefact that they require substantially fewer conjugation sites and loss ofbioactivity is minimized. Branched PEG molecules are described in U.S.Pat. No. 6,113,906, U.S. Pat. No. 5,919,455, U.S. Pat. No. 5,643,575,and U.S. Pat. No. 5,681,567, hereby incorporated by reference as iffully disclosed herein in their entirety.

The present invention further provides pharmaceutical compositionscomprising a pegylated compound according to the invention or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

The carrier must be “acceptable” in the sense of being compatible withthe active ingredient(s) of the formulation (and preferably, capable ofstabilizing peptides) and not deleterious to the subject to be treated.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy,for example as described in Remington: The Science and Practice ofPharmacy, A. R. Gennaro, ed., 20th edition, 2000. All methods includethe step of bringing the active ingredient(s) into association with thecarrier which constitutes one or more accessory ingredients.

Any suitable route of administration of the conjugates of the inventionto humans or for veterinary purposes is envisaged by the invention, forexample via conventional oral, intramuscular, intravenous, subcutaneous,intranasal and transdermal administration.

The invention further provides methods for treatment of diseases,disorders and conditions treatable by peptide and protein drugs whichcomprises the administration of a pegylated peptide or protein prodrugof the invention to an individual in need.

Thus, in one embodiment, there is provided a method for the treatment ofdiabetes mellitus or hyperglycemia which comprises administering to anindividual in need an effective amount of a pegylated FMS conjugate ofinsulin of or a HSA-Fmoc-insulin conjugate of the invention.

In another embodiment, there is provided a method for the treatment of aviral disease which comprises administering to an individual in need aneffective amount of a pegylated FMS conjugate of IFN-α2 of theinvention. The viral disease is particularly hepatitis B or hepatitis C.

In a further embodiment, the invention relates to a method for thetreatment of cancer such as bladder cancer, ovarian cancer, pancreaticcancer melanoma, non-Hodgkin's lymphoma, hairy cell leukemia, orAIDS-related Kaposi's sarcoma, which comprises administering to anindividual in need an effective amount of a pegylated FMS conjugate ofIFN-α2 of the invention.

In another embodiment, the invention relates to a method for reductionof food intake, treatment of obesity and diseases, conditions ordisorders which can be alleviated by reduction of food intake includinghypertension, dyslipidemia, cardiovascular risk, insulin-resistance, anddiabetes mellitus which comprises administering to an individual in needan effective amount of a pegylated FMS conjugate of PYY₃₋₃₆ of theinvention.

In another embodiment, there is provided a method for treatment ofchildren of pathologically short stature which comprises administeringto a child in need an effective amount of a pegylated FMS conjugate ofhGH of the invention.

In a further embodiment, the invention relates to an anti-agingtreatment which comprises administering to an individual in need aneffective amount of a pegylated FMS conjugate of hGH of the invention.

In still another embodiment, the invention provides a method fortreatment of a disease or disorder selected from the group consisting ofcardiovascular diseases, congestive heart failure, hypertension, acutekidney failure and adult respiratory distress syndrome (ARDS), whichcomprises administering to an individual in need an effective amount ofa pegylated FMS conjugate of ANP of the invention.

In yet another embodiment, the invention provides a method for treatmentof insulin-dependent diabetes mellitus, non-insulin-dependent diabetesmellitus, or gestational diabetes mellitus, or for prevention ofhyperglycemia which comprises administering to an individual in need aneffective amount of a pegylated FMS conjugate of exendin of theinvention.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES I. Chemical Section Pegylation Materials and Methods (ChemicalSection)

(i) Materials: Human (Zn²⁺-free) insulin was donated by Novo Nordisk(Bagsvaerd, Denmark) or by Biotechnology General (Rehovot, Israel), andwas used without further purification. Recombinant hGH was a gift fromBiotechnology General (Rehovot, Israel). Fmoc-Osu was obtained fromNovabiochem (Laüfelfingen, Switzerland). PEG₅-OSu (also referred hereinas PEG₅-OSu) and PEG₄₀-OSu (also referred herein as PEG_(40,000)-OSu)were from Shearwater (now Nektar Therapeutics, San Carlos, Calif., USA).TNBS, DTNB, α-lactalbumin, reduced glutathione (GSH), cystamine-2HCl,dithiothreitol (DTT), iodoacetamide, gentamicin and TPCK-treated trypsinwere purchased from Sigma Chemical Co. (St. Louis, Mo., USA).Non-glycosylated human IFN-α2 was prepared as described in WO 02/36067.Exendin-4, Peptide YY₃₋₃₆ and Peptide 27, a nonlysine-containingsynthetic peptide of 27 amino acids (AEISGQLSYVRDVNSWQHIWTNVSIEN) (SEQID NO: 14), used as control, were synthesized by the solid phase methodusing a multiple-peptide synthesizer (AMS 422, Abimed Analysen-TechnikGmbH, Langenfeld, Germany). All other reagents, including a long list ofcompounds used for synthesizing MAL-FMS-NHS, were of analytical gradeand purchased from Sigma Chemical Co. (St. Louis, Mo., USA).

(ii) Reverse-phase HPLC was performed with a Spectra-Physics SP8800liquid chromatography system (Spectro-Physics, San Jose, Calif.)equipped with an Applied Biosystem 757 variable wavelength absorbancedetector. The column effluents were monitored by UV absorbance at 220 nmand chromatograms were recorded on a chrom-Jet integrator(Thermo-Separation, Riviera Beach, Fla., USA). HPLC prepacked columnsused in the examples included LiChroCART 250-10 mm containing LiChrosorbRP-18 (7 μm) and LiChrospher 100 RP-18 (5 μm) 250-4 mm (Merck, Rathway,N.J., USA) and pre-packed Vydac RP-18 or RP-4 columns (22×250 mm; 12 μmbead size; Vydac, Hesperia, Calif., USA). Linear gradients were usedbetween solution A (0.1% TFA in H₂O) and solution B (0.1% TFA inacetonitrile-H₂O, 75:25). For analytical HPLC procedures, a lineargradient between 30 and 100% of solution B was run for 50 min at a flowrate of 0.8 ml/min.

(iii) HPLC analyses were performed using a Spectra-Physics SP8800 liquidchromatography system equipped with an Applied Biosystems 757 variablewavelength absorbance detector and a Spectra-SYSTEM P2000 liquidchromatography system equipped with a Spectra-SYSTEM AS100 auto-samplerand a Spectra-SYSTEM UV1000, all controlled by a ThermoQuestchromatograpy data system (ThermoQuest Inc., San Jose, Calif., USA). Thecolumn effluents were monitored by UV absorbance at 220 nm. AnalyticalRP-HPLC was performed using a prepacked Chromlith™ Performance RP-18e(4.6×100 mm, Merck KGaA, Darmstadt, Germany). The column was eluted witha binary gradient of 10-100% solution B over 10 min with a flow rate of3 ml/min (solution A was 0.1% TFA in H₂O and solution B was 0.1% TFA inacetonitrile:water, 3:1, v:v). Pegylated compounds were analyzed using aRP-4 column (250×4 mm, 5 μm bead size, VYDAC, Hesperia, Calif.) with abinary gradient of 10-100% solution B in 50 min at a flow rate of 1ml/min.

(iv) Mass spectroscopy: Mass spectra (MS) were determined usingmatrix-assisted laser-desorption/ionization-time-of-flight (MALDI-TOF)mass spectroscopy (Micromass UK Ltd.) and electrospray ionization massspectra (ESMS) techniques (Bruker-Reflex-Reflectron model, Germany, andVG-platform-II electrospray single quadrupole mass spectrometer,Micromass UK Ltd., respectively). The polypeptides were deposited on ametal target as cocrystals with sinaptic acid, and the mass spectrum wasdetermined in the positive ion mode.

(v) UV spectra: Ultraviolet spectra were obtained with a Beckman DU 7500spectrophotometer in 1 cm path length UV cuvettes.

(vi) Thin-layer chromatography was performed on silica-gel plates, thatwere developed either with chloroform:methanol:acetic acid (9.2:0.5:0.3,v:v:v, TLC, A) or by chloroform:methanol (9:1 v:v. TLC, B).

(vii) Amino acid analyses were performed following 6N HCl acidhydrolysis at 110° C. for 24 h using a Dionex Automatic amino acidanalyzer HP1090 (Palo Alto, Calif., USA). N-terminal sequence analyseswere performed with a Model 491A Procise Protein sequencer (AppliedBiosystems, Foster City, Calif., USA).

Identification of the intermediate compounds—Most of the chemicalcompounds used as reagents and intermediates in the Examples areidentified by their formulas in the Schemes 1-7 hereinafter and thefollowing characterization: the intermediates are identified by a boldunderlined letter a to k (small cap) or by the term Precursor and anumber in bold italics, i.e. Precursors 1-8.

Example 1 Synthesis of PEG_(5,000)-Fmoc-OSu (Precursor 1)

Precursor 1 of the formula depicted in Scheme 1 was prepared startingfrom 2-aminofluorene and t-Boc-alanine (BocAla) by several steps, asdepicted in Scheme 2.

1(a). Synthesis of 2-(t-BocAla-amino)fluorene (Intermediate a)

t-Boc-Ala (4.16 gr, 22 mmol) was dissolved in 11 ml dioxane.N,N′-Dicyclohexylcarbodiimide (DCC) (11 mmol in 11 ml 1 M DMF) was thenadded and the reaction was carried out for 3 hours at 25° C. understirring. Dicyclohexylurea (DCU) formed was removed by centrifugation.The symmetrical anhydride product thus obtained (1-Boc-Ala-anhydride)was reacted overnight under stirring with 2-aminofluorene (0.925 g, 11mmol) in 30 ml dioxane-water (1:1, v:v) containing 11 mmol of NaHCO₃.The white solid formed was collected and dried under P₂O₅ in vacuum for24 hours. Intermediate a was obtained in 60% yield (1.31 g, 3.34 mmol).It migrated on TLC (dichloromethane) with Rt=0.17. Mass-spectroscopyrevealed a mass of 352.45 Da (calculated mass=352.2 Da).

1(b). Synthesis of 9-formyl-2-(t-BocAla-amino)fluorene (Intermediate b)

Intermediate a (1.31 g, 3.34 mmol) obtained in Example 1(a) wasdissolved in 10 ml dry THF and combined with sodium hydride (NaH, 60%)(0.412 g, 11 mmol, 3.3 equivalents) suspended in dry THF. Ethyl formate(0.675 ml, 8.35 mmol) was then added and the reaction was carried outfor 1 hour under stirring and argon atmosphere. After addition of icechips and water, the organic solvent was removed by evaporation invacuum. The aqueous solution was washed with ether and acidified to pH5.0 with acetic acid. The precipitate formed was dissolved in ethylacetate, the organic solution was washed several times with 0.5 MNaHCO₃, and dried over anhydrous sodium sulfate. The yellow solid formedafter evaporation was triturated with ether and dried in vacuum.Intermediate b was obtained in 35% yield (0.46 g, 1.2 mmol). It migratedon TLC (chloroform:methanol:acetic acid, 9.2:0.5:0.3, v:v:v) withRf=0.59. Mass-spectrum analysis (electrospray ionization technique)revealed a mass of 380.26 Da (calculated mass=380.2 Da).

1(c). Synthesis of 9-hydroxymethyl-2-(t-BocAla-amino) fluorene(Intermediate c)

Intermediate b (0.46 g, 1.2 mmol) obtained in Example 1(b) was suspendedin dry methanol. Solid sodium borohydride (NaBH₄) (57 mg, 1.5 mmol) wasadded in several aliquots and the reaction mixture was stirred for 4hours. The crude product obtained was further purified by flashchromatography on silica gel column and eluted with chloroform:methanol(95:5), to yield 80 mg (17%, 0.2 mmol) of pure Intermediate c thatmigrated on TLC (chloroform:methanol; 9:1, v:v) with Rf=0.53. Massspectrum analysis revealed a mass of 382.2 Da (calculated mass=382 Da).¹H-NMR (CD₃SOCD₃) δ: 1.4 (S, 12H), 3.8-4.0 (m, 2H), 4.29 (d, 1H), 6.2(s, broad), 7.2-7.3 (m, 2H), 7.6-8.0 (m, 4H), 8.4 (5, 1H).

1(d). Synthesis of 9-hydroxymethyl-2-(Ala-amino)fluorene (Intermediated)

Intermediate c (80 mg, 0.2 mmol) obtained in Example 1(c) was dissolvedin 5 ml of dichloromethane:trifluoroacetic acid (TFA) (1:1, v:v). Afterone hour, the solvents were removed by evaporation, and Intermediate dwas suspended in ether, collected by precipitation and lyophilized.

1(e). Synthesis of Intermediate e

To a solution of Intermediate d (0.2 mmol) obtained in Example 1(d) andNaHCO₃ (0.8 mmol) in 3 ml H₂O, PEG_(5,000)-OSU (1 g, 0.2 mmol) wasadded. The reaction was carried out at 25° C. for several hours, withstirring. Product formation was verified by analytical HPLC procedureusing C18 column (Rt=36.6 min). Mass spectrum analysis (MALDI) ofIntermediate e revealed a mass of 5342 Da.

1(f). Synthesis of PEG_(5,000)-Fmoc-OSu (Precursor 1)

To a solution of the Intermediate e obtained in Example 1(e) (0.2 mmol)in 2 ml chloroform, triphosgene (1 mmol, 5 molar excess) in 3 ml coldchloroform was added portionwise. Reaction was carried out overnightwith stirring. The solvent was then evaporated and the residue wasdissolved in 1.0 ml dry THF. N-hydroxysuccinimide (NHS) (100 mg, 4equivalents) and 2,4,6-trimethylpyridine (0.163 ml, 6 equivalents) wereadded, and the reaction was carried out for 2 hours at room temperaturewith stirring. The title product, PEG_(5,000)-Fmoc-OSu, was purified tohomogeneity by preparative HPLC procedure using a C18 column (HPLC,Rt=39.0 min.). Mass spectrum analysis revealed a mass of 5654 kDa.

Unlike Fmoc-OSu, PEG_(5,000)-Fmoc-OSu is highly soluble in aqueoussolutions, and absorbs in the UV region with a molar extinctioncoefficient of ε₂₈₀=21,200 and ε₃₀₁=10,100.

Example 2 Synthesis of PEG_(5,000)-FMS-OSu (Precursor 2)

Precursor 2, PEG_(5,000)-FMS-OSu, depicted in Scheme 1, was prepared bysulfonation of Precursor 1, PEG_(5,000)-Fmoc-OSu, with chlorosulfonicacid, as depicted in Scheme 2. Briefly, to a solution of Precursor 1 in4.0 ml CH₂Cl₂, cooled to 0° C., a solution of ClSO₃H in CH₂Cl₂ was addeddropwise over a period of 15 min and the solution was stirred for 2hours at 25° C. The product, PEG_(5,000)-FMS-OSu, sulfonated at position2 of the fluorene ring, was purified by preparative HPLC-procedure, andcharacterized by mass spectroscopy, elementary analysis (for sulfur),following extensive dialysis, and for its rate of hydrolysis (at pH 8.5,37° C.) following conjugation to either gentamicin or insulin.

Example 3 Synthesis of PEG₄₀₀₀₀-Fmoc-OSu (Precursor 3)—

Precursor 3, PEG₄₀₀₀₀-Fmoc-OSu, depicted in Scheme 1, was prepared asdescribed in Example 1, steps 1(e) and 1(f), but replacing PEG₅₀₀₀-OSuwith PEG₄₀₀₀₀-OSu under the same reaction conditions.

Example 4 Synthesis of PEG₄₀₀₀₀-FMS-OSu (Precursor 4)

Precursor 4, PEG₄₀₀₀₀-FMS-OSu, depicted in Scheme 1, was prepared bysulfonation of Precursor 3, PEG₄₀₀₀₀-Fmoc-OSu, as described in Example2, but replacing Precursor 1 with Precursor 3 under the same reactionconditions.

Example 5 “One-Step” Procedure for Preparation of PEG-Fmoc/DrugConjugates and PEG-FMS-Drug Conjugates

For the preparation of PEG-Fmoc and PEG-FMS conjugates with drugsaccording to the invention, a “one-step” procedure can be used wherein aPEG-Fmoc-OSu or PEG-FMS-OSu precursor reacts with one or more aminogroups of the drug in aqueous conditions.

Thus, in the following Examples 6-8, solid Precursor 1, PEG₅₀₀₀-FMS-OSu,obtained in Example 1, was added at a 10-fold molar excess to stirredsolutions of gentamicin or insulin (10 mg/ml) in phosphate-bufferedsaline (PBS) buffer, pH 7.4, at 0° C. Under these conditions, five toseven moles of Precursor 1 were incorporated per mole protein. Thereaction was completed within 15 minutes after addition of the solidPEG-Fmoc-OSu.

Example 6 Synthesis of (PEG₅₀₀₀-Fmoc)₁-gentamicin

Solid Precursor 1 obtained in Example 1 (6 mg, 1 mmol) was added to astirred solution of gentamicin (200 μmol in 1.0 ml 0.01 M NaHCO₃, pH˜7.5). The reaction was carried out for 3 hours at 25° C. and thendialyzed against H₂O at 7° C. for several days. Under these dialyzingconditions, free gentamicin, that has not been covalently linked toPEG-Fmoc, was dialyzed out. The product, PEG₅₀₀₀-Fmoc-gentamicin,contained one mol PEG-Fmoc covalently linked to one mol gentamicin asjudged by its absorbance at 280 nm, and by amino acid analysis,following acid hydrolysis of a measured aliquot. The hydrolyzatecontained alanine (derived from the PEG-ala-Fmoc moiety) and two peaksthat emerged at the positions of proline and leucine, following acidhydrolysis of gentamicin (not shown).

Example 7 Synthesis of (PEG₅₀₀₀-Fmoc)-2-gentamicin

Solid Precursor 1 (11.3 mg, 2.1 μmol) was added to a stirred solution ofgentamicin (0.5 mg, 1 μmol, in 1.0 ml 0.05 M NaHCO₃). The reaction wascarried out for 2 hours at 25° C. and dialyzed overnight. The product,(PEG₅₀₀₀-Fmoc)-2-gentamicin, contained about two moles PEG-Fmoccovalently linked to gentamicin, as verified by amino acid analysisfollowing acid hydrolysis (see Example 6 above) and by determining theamount of non-modified amino groups with TNBS.

Example 8 Synthesis of (PEG₅₀₀₀-Fmoc)-1-Insulin and(PEG₅₀₀₀-Fmoc)-2-Insulin

For the preparation of (PEG₅₀₀₀-Fmoc)₁-insulin, to a stirred solution ofZn²⁺-free insulin (1 mg in 2.0 ml 0.01 M NaHCO₃, 0.172 μmol), a freshsolution of Precursor 1 (8.8 μl, 20 mg/ml in DMF) was added (1.76 mg,0.329 μmol, 1.9 molar excess of reagent over the protein). The reactionwas carried out for 2 hours at 25° C. The reaction mixture was dialyzedovernight against H₂O, to remove NaHCO₃ and DMF. The title productcontained about one mol PEG-Fmoc per mol insulin, as judged by severalprocedures described in the Biological Section hereinafter. Theconcentration of insulin in the sample was routinely determined by acidhydrolysis of a 20 μl aliquot, followed by amino acid analysis, and wascalculated according to glutamic acid (7 residues), aspartic acid (3residues) and isoleucine (2 residues).

The preparation of (PEG₅₀₀₀-Fmoc)-2-insulin was carried out as describedabove while using 4.63 mg, 0.865 μmol, 5 molar excess of reagent overthe protein.

Example 9 Synthesis ofN-[2-(3-acetylthiopropionylamino)-9-fluorenyl-methoxycarbonyloxy]-succinimide(Precursor 5)

Precursor 5, depicted in Scheme 3, was synthesized starting from2-aminofluorene by the procedure depicted in Scheme 4, as follows:

9(a). Synthesis of 2-(t-Boe-amino)fluorene (Intermediate f)

Di-t-butyl-dicarbonate (Boc anhydride, 14 g, 64.2 mmol) solution indioxane (50 ml) was combined with 2-aminofluorene (10 g, 55 mmol in 100ml dioxane:water, 1:1, v:v) and with NaHCO₃ (9.24 g, 110 mmol), andstirred overnight. The white solid thus formed was filtered, washed withice water (200 ml) and dried under high vacuum. Intermediate f wasobtained in 60% yield (9.24 g, 33 mmol. TLC (chloroform:methanol:aceticacid, 9.5:0.5:0.3, v:v:v); Rf=0.73. Calculated ESMS=280.4 Da, foundESMS=280.45 Da.

9(b). Synthesis of 9-formyl-2-(1-Boc-amino)fluorene (Intermediate g)

Intermediate f (3 g, 10 mmol), obtained in step 9(a), was dissolved indry THF (30 ml) and added to a suspension of sodium hydride (NaH) (60%,1.23 g, 33 mmol, 3.3 eq) in dry THF under Argon atmosphere. Ethylformate (2 ml, 25 mmol, 2.5 eq) was then added and the reaction mixturewas stirred for 1 hour. Ice chips and water were added, the organicsolvent was evaporated, and the aqueous solution was washed with etherand acidified with acetic acid (pH 5). The precipitate thus formed wasdissolved in ethyl acetate, washed with NaHCO₃ (0.5 N), brine and driedover anhydrous sodium sulfate. The yellow solid was washed with etherand dried. Intermediate g was obtained in 90% yield (2.8 g, 9 mmol). TLC(chloroform:methanol:acetic acid; 9.5:0.5:0.3, v:v:v), Rf=0.66.Calculated ESMS=309 Da, found ESMS=309.2 Da. M−1: 308.20, M+Na: 332.36,dimer [M+Na]⁺: 641.59.

9(c) Synthesis of 9-hydroxymethyl-2-(1-Boc-amino)fluorene(Intermediateh)

Sodium borohydride (NaBH₄) (0.38 g, 10 mmol) was added portionwise to asuspension of Intermediate g (2.8 g, 9 mmol) obtained in step 9(b), indry methanol, and the reaction allowed to proceed for 4 hours. Theproduct, Intermediate h, was purified by flash chromatography on silicagel column, that was eluted with chloroform:methanol (98:2, v:v), andwas obtained in 50% yield (1.4 g, 4.5 mmol). TLC (chloroform:methanol,9:1, v:v), Rf=0.54; Calculated ESMS=311 Da, found ESMS=311.42 Da, for[M+Na]⁺=334.42, for [M-K]-=350.34.

9(d). Synthesis of 9-hydroxymethyl-2-aminofluorene (Intermediate i)

Intermediate h (1.4 g, 4.5 mmol) obtained in step 9(c) was dissolved in110 ml 5N HCl in dioxane. After 1 hour, the solvent was concentrated byevaporation and the product was precipitated with ether and lyophilized.Intermediate i was obtained in 84%, yield (0.79 g, 3.78 mmol). TLC(chloroform:methanol, 9:1, v:v), Rf=0.38; calculated ESMS=211.26 Da,found ESMS for [M+H]+=211.10 Da.

9(e). Synthesis of 3-S-acetylthiopropionic acid

Pyridine (6.9 ml, 84.6 mmol) was added to a mixture of3-mercaptopropionic acid (2.5 ml, 28.2 mmol) and acetic anhydride (16ml, 84.6 mmol). The reaction solution was stirred for 16 h andconcentrated by vacuum. Water (5 ml) was added for 20 min and thesolution was concentrated by vacuum. The obtained oil was dissolved inether (50 ml) and washed with water and KHSO₄ (0.5 N). The etherfraction was dried with Na₂SO₄ and vacuum to produce the title product(Yield: 85%, 24 mmol, 3.6 g). ¹H-NMR (CDCl₃) δ: 2.21 (s, 3H), 2.6 (t,2H), 2.9 (t, 2H).

9(f). Synthesis of 3-S-acetylthiopropionic anhydride

3-S-acetylthiopropionic acid of step 9(e) (1.8 g, 12 mmol) and DCC (1.4g, 6 mmol) were dissolved in DMF (15 ml) for 4 hours. The precipitatedDCU was filtered out and the anhydride thus formed was kept at 4° C.until used.

9(g). Synthesis of9-hydroxymethyl-2-(3-acetylthiopropionyl-amino)-fluorene (Intermediatej)

Intermediate i (0.422 g, 2 mmol) obtained in step 9(d) and NaHCO₃ (0.74g, 18 mmol) were dissolved in water/dioxane (1:1, 20 ml) and3-S-acetylthiopropionic anhydride of step 9(f) in DMF (17 ml, 6 mmol)was added. The reaction solution was stirred for 1 hour. The organicsolvents were removed by vacuum and the liquid solution was extractedwith ether, washed with Na₂SO₄ (0.5 N) and water and dried by vacuum.The obtained crude product, Intermediate j, was further purified byflash chromatography on silica gel column and elution with ethylacetate:hexane (1:1, v:v). Yield: 60%, 0.4 g, 1.2 mmol. ESMS, (ca. 431)M+Na⁺: 464, dimer 2M: 682, dimer 2M: Na⁺: 705.67. ¹H-NMR (CDCl₃)₆: HPLC(Chromolith column) 5.3 min (10-100% solution B in 10 min, 3 ml/min).

9(h). Synthesis of Precursor 5

Intermediate j obtained in step 9(g) was reacted withN-hydroxysuccinimide and phosgene, as depicted in Scheme 4. Thus,pyridine (0.215 ml, 2.7 mmol) was added dropwise to a stirred solutionof Intermediate j (0.385 g, 0.9 mmol) and triphosgene (0.265 g, 0.9mmol, 3 eq) in dry THF (5 ml). After 20 min, the precipitated pyridinehydrochloride salt was filtered out and the THF was removed byevaporation. The obtained oil was dissolved in dry THF (10 ml).N-hydroxysuccinimide (0.5 g, 4.4 mmol, 5 eq) and pyridine (0.215 ml, 2.7mmol) were then added and the solution stirred for 20 min. Theprecipitated pyridine hydrochloride salt was filtered out and the THFwas removed by vacuum. The obtained oil was further purified by flashchromatography on silica gel column and elution with ethylacetate:hexane (1:1 and 4:1). The product, Precursor 5, was obtained ina yield of 88% (0.38 g, 0.78 mmol). ESMS: (ca 482) M: 482.2. HPLC(Chromolith column) 6.68 min (10-100% solution B in 10 min, 3 ml/min).TLC (ethyl acetate:hexane, 1:1, v:v) Rf=0.4.

Example 10 Synthesis ofN-[2-(3-acetylthiopropionylamino)-7-sulfo-9-fluorenylmethoxycarbonyloxy]-succinimide(Precursor 6)

Precursor 6, depicted in Scheme 3, was prepared by sulfonation ofPrecursor 5 with chlorosulfonic acid as described above in Example 2.

Example 11 Synthesis of MAL-Fmoc-NHS, N-[2-(maleimidopropionylamino)fluoren-9-yl-methoxycarbonyloxy[succinimide) (Precursor 7)

Precursor 7, MAL-Fmoc-NHS depicted in Scheme 3, was prepared as depictedin Scheme 5 starting from 2-aminofluorene through the synthesis of theIntermediates f to i, described in Example 9 above, followed by thesteps below:

11(a). Synthesis of 9-Hydroxymethyl-2-(maleimidopropionyl amino)fluorene(Intermiediate k)

3-Maleimidopropionic anhydride was prepared by dissolving3-maleimido-propionic acid (1 g, 5.9 mmol) and DCC (0.69 g, 2.95 mmol)in 10(ml DMF and incubating for 4 h. DCU was filtered out and theanhydride thus formed was kept at 4° C.

9-Hydroxymethyl-2-amino-fluorene (Intermediate i; (0.4 g, 1.9 mmol) andNaHCO₃ (0.74 g, 8.85 mmol) were dissolved in water and3-maleimidopropionic anhydride in DMF (12 ml, 2.95 mmol) was added. Thereaction mixture was stirred for 40 min and product formation monitoredby analytical HPLC on a Chromolith column (Rt=4.46 min, 10-100% B in 10min, 3 ml/min). The crude product, Intermediate k, was purified usingpreparative HPLC (RP-18 column, 10-100% acetonitril:water [75:25; v:v],60 min, 12 ml/min). Yield: 57%, 1.08 mmol, 0.39 g. CalculatedESMS=362.38 Da, found ESMS=362.42 Da).

11(b). Synthesis of MAL-FMOC-NHS

Pyridine (0.167 ml, 2 mmol) was added dropwise to a stirred solution ofIntermediate k (0.37 g, 1.02 mmol) obtained in step 11(a) andtriphosgene (0.425 g, 1.43 mmol, 4.2 eq) in dry THF (10 ml). After 20min the precipitated pyridine hydrochloride salt was filtered out andthe THF removed by vacuum. The oil obtained was dissolved in 10 ml dryTHF with N-hydroxysuccinimide (0.61 g, 5.3 mmol). Pyridine (0.26 ml, 3.2mmol) was then added and the solution was stirred for 20 min. Someadditional precipitated pyridine hydrochloride salt was filtered out andthe THF removed by vacuum. The oil obtained was dissolved in chloroform(100 ml) and washed with an aqueous NaHCO₃ solution (0.5 N, 3×50 ml),HCl (0.1 N, 3×50 ml), water (2×50 ml) and brine. The chloroform wasremoved by vacuum and the product, Precursor 7, was desiccated. Yield:89% (0.9 mmol, 0.45 g). HPLC (Chromolith column) Rt=5.7 min (10-100% Bin 10 min, 3 ml/min). Calculated ESMS=503 Da, found ESMS for[M+Na]⁺=526.38 Da, found ESMS for [M+K]⁺=542.30 Da.

Example 12 Synthesis of MAL-FMS-NHS, N-[2-(maleimido-propionylamino)7-sulfo-fluoren-9-yl]methoxycarbonyloxysuccinimide (Precursor 8)

Precursor 8, depicted in Scheme 3, herein also identified by theabbreviation MAL-FMS-NHS (or MAL-FMS-OSu), was prepared by sulfonationof Precursor 7, MAL-Fmoc-NHS, with chlorosulfonic acid as depicted inScheme 5, as follows:

To a solution of Precursor 7 (0.2 mmol, 0.1 g) obtained in Example 11 intrifluoroacetic acid (TFA) (10 ml), chlorosulfonic acid (0.5 ml) wasadded. After 15 minutes, cold ether (90 ml) was added and theprecipitated product. Precursor 8, was washed with ether (×3) anddesiccated. Yield: 95% (0.11 g, 0.19 mmol). HPLC (Chromolith column)Rt=2.65 min (10-100% B in 10 min. 3 ml/min). Calculated ESMS=583 Da,found ESMS for [M−H]+=582.24 Da, for [M+H]⁺=584.52 Da, and for[M+Na]⁺=606.47 Da.

Example 13 Chemical Characterization of MAL-FMS-NHS

MAL-FMS-NHS or MAL-FMS-OSu is a water-soluble, hetero-bifunctionalreagent consisting of a sulfonated fluorenylmethoxycarbonylN-hydroxy-succinimide ester that reacts with peptide and protein aminogroups. A maleimide group was attached to the fluorenyl backbone toenable coupling to sulfhydryl-containing PEG₄₀₀₀₀-SH.

MAL-FMS-NHS is a cleavable reagent capable of reacting covalently withthe amino side chains of peptides, proteins and aminoglycosides.According to the present invention, it enables PEG chains to be linkedto peptides and proteins through a slowly hydrolysable chemical bond.PEG-FMS-peptide/protein conjugates thus formed undergo spontaneoushydrolysis at a slow rate upon incubation at ph 8.5, 37° C., with at_(1/2) value of 8-14±2 h, generating the unmodified parent molecule.

Chemical features of MAL-FMS-NHS. Table 1 summarizes several of thecharacteristic features of MAL-FMS-NHS. It is a water- and DMF-solublecompound. Mass spectrum analysis has yielded a calculated mass of 583Da. The compound absorbs in the U.V. region with a molar extinctioncoefficient ε₂₈₀=21,000±200 mol⁻¹ cm⁻¹. The curve shows a maximum at 290nm, and a shoulder extending up to 330 nm. At 320 nm, where peptides orproteins absorb negligibly, MAL-FMS-NHS, either free or covalently boundto proteins, absorbs with a molar extinction coefficient ε₃₂₀=16,100±150mol⁻¹ cm⁻¹ (Table 1).

Stability of the MAL-functional moiety in aqueous solutions. In order totest the stability of the maleimide function of MAL-FMS-NHS in aqueoussolutions at different pH values, MAL-FMS-NHS (1 mM) was incubated atroom temperature in water (pH 6.0), in 0.007M acetic acid (pH˜4.0), in0.1M phosphate buffer (pH 7.4), and in 0.1M NaHCO₃ (pH 8.5). At severaltime points, aliquots were allowed to react with a slight excess ofreduced glutathione (GSH) (15 min at pH 7.2) and the concentration ofunreacted GSH was determined with DTNB (5,5′-dithio-bis-2-nitrobenzoicacid. Ellman's reagent), by measuring the absorbance of the producedyellow colored 5-thio-2-nitrobenzoic acid (TNB) at 405 or 412 nm. FIG. 1demonstrates the potency of the reaction of MAL-FMS-NHS with GSH as afunction of time, at different pH values. MAL-FMS-NHS was found fullystable at pH 4 to 6 over a period of a day or two and at pH 7.2 it isstable over a period of several hours. At pH 8.5, the maleimide moietywas destroyed at an accelerated rate (t_(1/2) value=2.7±0.3 hr, FIG. 1).

Example 14 Degree of Incorporation of MAL-FMS-NHS at pH 7.2 intoα-Lactalbumin as a Function of the Amount of Added Reagent

To evaluate the degree of incorporation of the maleimide moiety intopeptides/proteins as a function of the reagent, α-lactalbumin was usedas a model. Thus, to samples of α-lactalbumin (α-LA, 1.0 ml of 1 mg/ml),MAL-FMS-NHS was added in 0.1M PBS, pH 7.2, at concentrations rangingfrom 1 equivalent up to 14 molar equivalents of MAL-FMS-NHS, for 30 minat 25° C., that is, under experimental conditions where themaleimide-alkylating capacity of the spacer remains unmodified. For eachtreatment, the amount incorporated into the protein was determined by UVabsorbance at 280 nm, after dialysis, and by quantitating the amount ofunmodified amino side-chain moieties with TNBS. As shown in FIG. 2,about one mole of MAL-FMS was incorporated into α-LA per 2.1 mole ofreagent added, up to a fourteen molar excess over the protein.

Example 15 Synthesis of PEG₄O—SH

PEG₄₀-SH was prepared either by reaction of 2-tritylthioethylamine andPEG₄₀-OSu as described below in 15(a), or from PEG₄₀-OSu and cystamine[2,2′-dithio-bis(ethylamine)] dihydrochloride, as described below in 15(b).

15(a). Synthesis of PEG₄₀-SH from 2-tritylthioethylamine and PEG₄₀-OSu

Synthesis of 2-tritylthioethylamine. 2-Amino-ethanethiol (3 g, 26.5mmol) and triphenylmethyl alcohol (7 g, 26.9 mmol) were dissolved in TFA(20 ml) and stirred for 30 min at room temperature. The TFA was removedby evaporation and the remaining oil residue was dissolved in ether (400ml) and stored at −20° C. The product isolated was washed with cooledether and water. A concentrated ammonia solution (25%, 30 ml) was thenadded, and the aqueous phase was washed several times with ether. Theether was removed by vacuum and the product was desiccated. Yield, 78%(6.6 g, 20.7 mmol), Rt=5.35 min.

2-Tritylthioethylamine and PEG₄₀-OSu (0.25 g, 6.25 μmol) were dissolvedin 4 ml THF:acetonitrile (1:2, v:v). After one hour, the product wasprecipitated with cooled ether and washed (×3). The dry pellet wasdissolved in TFA:triethylsilane:water (95:2.5:2.5, 6 ml) and, after onehour, PEG₄₀-SH was precipitated by cooled ether (100 ml) and desiccated.

15(b). Synthesis of PEG₄(rSH from Cystamine and PEG₄₀-OSu

PEG₄₀-OSu was dissolved at a concentration of 40 mg/ml in an aqueoussolution of cystamine-di-HCl (1M) and brought to pH 8.5 with NaHCO₃.Reaction was carried out for 2 h at 25° C. The product thus obtained wasdialyzed overnight against 0.1 M NaHCO₃, treated with 30 mMdithiothreitol (25° C., 1 h) and re-dialyzed against 0.01 M HClcontaining 10 mM ascorbic acid. PEG₄₀-SH was obtained with a 93% yield.It contained 1 mole sulfhydryl moiety per mole PEG₄₀, as determined withDTNB in the presence of ascorbate. PEG₄₀-SH was kept frozen until used.

Example 16 Preparation of PEG-FMS-Drug Conjugates from MAL-FMS-NHSProcedure I

For the preparation of PEG-FMS-drug conjugates according to theinvention, a “two-step” Procedure I can be used as depicted in Scheme 6whereby MAL-FMS-NHS is first reacted with an amino-containing drug orwith the amino group of a peptide or protein drug and the resultingMAL-FMS-drug conjugate is further reacted with PEG-SH. as follows:

16(a). Preparation of MAL-FMS-Drug Conjugates

In the first step, a MAL-FMS-drug conjugate is prepared by adding one toseven molar excess of MAL-FMS-NHS, Precursor 8, to a stirred solution ofthe drug (1 mg/ml) in 0.1 M PBS buffer, pH 7.2. The reaction is carriedout for 30 min at 25° C., and then dialyzed against water at 7° C. overa period of 24 h to remove excess MAL-FMS-NHS. The amount of FMS-MALresidues incorporated into the drug, e.g. a peptide or protein drug, isdetermined both by the absorption at 320 nm using molar extinctioncoefficient of ε₃₂₀=16,100, and by quantitating the incorporated MALfunction into the drug. This is performed by adding a measured aliquotinto a GSH solution (0.1 mM in phosphate buffer, pH 7.2). Theconcentration of unreacted GSH remained is determined with DTNB.

16(b). Preparation of PEG-FMS-Drug Conjugates

In the second step, a stoichiometric amount of solid PEG₄₀-SH is addedto the MAL-FMS-drug conjugate of step 16(a), and the reaction proceedsfor additional 60 min in 0.1 M phosphate buffer, pH 7.2, containing 2 mMascorbic acid. The PEG-FMS-drug conjugate thus obtained is furtherpurified by HPLC-procedures using C4 or C18 reverse phase columns underconditions resolving the conjugate from the drug, preferably a peptideor protein drug (or protein-FMS-MAL) that has not been covalently linkedto PEG₄₀-SH.

Example 17 Preparation of PEG-FMS-Drug Conjugates from MAL-FMS-NHSProcedure II

PEG-FMS-drug conjugates according to the invention may be also preparedby an alternative “two-step” Procedure II depicted in Scheme 6 wherebyMAL-FMS-NHS is first reacted with PEG-SH and the resulting PEG-FMS-NI—ISconjugate is further reacted with an amino-containing drug or with theamino group of a peptide or protein drug. as follows:

17(a). Preparation of PEG-FMS-NHS Conjugate

In the first step, a PEG-FMS-NHS conjugate is prepared by MAL-FMS-NHS toa stirred solution of PEG-SH in PBS buffer, pH 7.4. The reaction iscarried out for 30 min at 25° C. and the product is then purified byHPLC and lyophilized.

17(b). Preparation of PEG-FMS-Drug Conjugates

In the second step, a stoichiometric amount of the MAL-FMS-NHS conjugateof step 17(a) is added to the amino-containing drug, and the reactionproceeds under stirring for 2 hours. The product is then purified byHPLC and lyophilized.

Example 18 Synthesis of (PEG₄₀-FMS)₁-Insulin

(PEG₄₀-FMS)₁-insulin, was prepared by the two-step Procedure I describedin Example 16. Thus, Mal-FMS-NHS (6 g, 10 μmol) was dissolved in water(0.25 ml) and added to an insulin (Zn²⁺-free, 25 mg, 4.16 μmol) solutionin PBS (0.5 ml, pH=7.4). The pH was adjusted to 7-8 with NaHCO₃. Thereaction was stopped after 30 min with diluted hydrochloric solution(pH=6). The product was isolated using preparative HPLC (C18 column,10-80% acetonitrile:water, 75:25, v:v, 50 min, 10 ml/min) and identifiedas monosubstituted insulin derivative Mal-FMS-insulin using electrospraymass spectrometry (ESMS (ca. 6280), M−1: 6278.83, M+1: 6280.73).Analytical HPLC (Chromolith column), Rt=4.58 min.

PEG₄₀-SH (20 mg, 0.5 μmol) was added to a solution of theMal-FMS-insulin intermediate (2 mg, 0.317 μmol) in PBS (0.3 ml, pH=7.4),the pH was adjusted to 7-8 with NaHCO₃ and the reaction was stoppedafter 30 min with diluted hydrochloric solution (pH=6). The titleproduct, PEG₄₀-FMS-insulin, was subjected to analytical HPLC (Chromolithcolumn), Rt=6.6 min.

Example 19 Synthesis of PEG₄₀-FMS-exendin-4

PEG₄₀-FMS-exendin-4 was prepared by the two-step Procedure I describedin Example 16. Thus, 280 μg of MAL-FMS-NHS (28 l from a fresh solutionof MAL-FMS-NHS in DMF, 10 mg/ml, 2.0 molar excess over the peptide) wasadded to a stirred solution of exendin-4 (1 mg in 1.0 ml 0.1 M phosphatebuffer, 2 mM ascorbate, pH 7.2, 0.239 mM). After 7 min, solidPEG₄₀₀₀₀-SH was added to a final concentration of 0.5 mM (2.1 molarexcess over the peptide). The reaction was carried out for 1 h hour,dialyzed overnight against water and then further filtered throughCentricon with a molecular weight cutoff of 50 kDa to remove anyresidual exendin-4 or MAL-FMS-exendin-4 that had not linked toPEG₄₀₀₀₀-SH. The concentration of the conjugate was determined by acidhydrolysis of a 20 μl aliquot followed by amino acid analysis, accordingto aspartic acid (2 residues), alanine (2 residues) and valine (1residue), and absorbance at 280 nm was monitored. The calculatedextinction coefficient at 280 nm is 26940±100, a value that is theadditive absorbance of exendin-4 (ε₂₈₀=5740) and PEG₄₀₀₀-FMS(ε₂₈₀=21200). A solution of 20 μM PEG₄₀₀₀₀-FMS-exendin had OD₂₈₀=0.51.Thus the conjugate had a 1:1 PEG₄₀₀₀₀-FMS/exendin stoichiometry (asdetermined from the UV absorption of the conjugate followingpurification and acid hydrolysis followed by amino acid analysis asdescribed above).

Example 20 Synthesis of PEG₅₀₀₀-FMS-exendin-4

PEG₅₀₀₀-FMS-exendin-4 was prepared by the two-step Procedure IIdescribed in Example 17. Thus, MAL-FMS-MHS (1.8 mg, 3 mmol) was added toa solution of PEG₅₀₀₀-SH (15 mg, ˜3 μmol) in PBS (0.5 ml, pH=7.4) andthe reaction solution was stirred for 30 min. The productPEG₅₀₀₀-FMS-NHS was purified by HPLC on a RP-4 column and lyophilized.

PEG₅₀₀₀-FMS-NHS (2.3 mg, ˜0.42 μmol) was then added to a solution ofexendin-4 (1.6 mg, 0.4 μmol) in PBS (0.5 ml, pH=8) and the reactionsolution was stirred for 2 h. The product PEG₅₀₀₀-FMS-exendin-4 waspurified by HPLC on a RP-4 column and lyophilized.

Example 21 Synthesis and Characterization of PEG₄₀-FMS-IFNα2

The attachment of a single PEG-chain of 40 kDa to IFNα2 appearssufficient to grossly arrest kidney filtration of the conjugate (Bailonet al., 2001). We therefore envisioned that the linkage of a singlePEG₄₀-FMS chain to IFNα2 would suffice to obtain a prolonged-actingconjugate that releases IFNα2, with a desirable pharmacokinetic profile.In the procedure found most optimal for introducing one molePEG₄₀-FMS/mol protein, IFNα2 was allowed to react first with 3equivalents of MAL-FMS-NHS, at pH 7.2, for 7 min. followed by theaddition of 3 equivalents of PEG₄O—SH, at pH 7.2, for 1 hour. The NHSfunction of MAL-FMS-NHS is relatively unstable at prolonged aqueousneutral conditions, whereas the MAL function of the spacer preserves itsalkylating capacity for several hours at pH 7.2 (not shown). Wetherefore preferred to use Procedure I of Example 16 and to reactMAL-FMS-NHS first with IFNα2 and to subsequently link PEG₄₀-SH to theIFNα2-FMS-MAL conjugate.

To a stirred solution of IFNα2 (1 mg/1.0 ml) in phosphate buffer, pH 7.2(52 μM), 91 μg of MAL-FMS-NHS was added (9.1 μl from a fresh solution ofMAL-FMS-NHS, (10 mg/ml) in DMF, (3.0 molar excess over the protein).After 7 min, PEG₄₀-SH was added to obtain a final concentration of 156μM (three molar excess over the protein). The reaction was carried outfor 1 h, and then dialyzed overnight against H₂O to remove residual DMFand phosphate buffer. The conjugate thus obtained was characterized byMALDI-TOF as PEG₄₀-FMS-IFN-α2.

Table 2 summarizes several characteristic features of the conjugate thusobtained. MALDI-TOF mass spectra analysis shows a 1:1 PEG₄O-FMS/IFNα2stoichiometry. The experimental mass obtained, 63540 Da, corresponds tothe additive masses found for PEG₄₀-SH (43818 Da), IFNα2 (19278 Da) andof the spacer molecule following conjugation (473 Da). PEG₄₀-FMS-IFNα2migrates on analytical HPLC as a wide peak with Rt value=43 min. Theconjugate is highly soluble in aqueous solutions. It has a molarextinction coefficient 628=39270±100, corresponding to the absorption ofthe native cytokine and of FMS (ε₂₈₀=21,200) (Gershonov et al., 2000).

Example 22 Synthesis of (PEG₄₀-FMS)₂—IFNα2

(PEG₄₀-FMS)₂—IFNα2 was prepared as described in Example 21 above forPEG₄₀-FMS-INFα2, but using 6 eq (16 μg, 44.5 nmol, in 10 μl DMF) ofMAL-FMS-NHS.

Example 23 Synthesis of PEG₄₀-FMS-PYY₃₋₃₆

In the procedure found most optimal for coupling about one mole ofPEG₄₀-SH per mol of MAL-FMS-PYY₃₋₃₆, PYY₃₋₃₆ was allowed to react firstwith 2 equivalents of MAL-FMS-NHS, at pH 7.2, for 7 min, followed by theaddition of 2.1 equivalents of PEG₄₀-SH, at pH 7.2, for 1 hour.

To a stirred solution of PYY₃₋₃₆ (1 mg in 1.0 ml phosphate buffer, pH7.2, 10 mM Na ascorbate (0.247 μM), 288 μg of MAL-FMS-NHS was added(28.8 μl, from a fresh 10 mg/ml solution of MAL-FMS-NHS in DMF; twofoldmolar excess over the peptide). After 7 min, PEG₄₀-SH was added toobtain a final concentration of 0.52 mM (2.1 molar excess over thepeptide). The reaction was carried out for 1 h and the mixture was thendialyzed overnight against water. The resulting PEG₄₀-FMS-PYY₃₋₃₆ wascharacterized by MALDI-TOF MS.

Example 24 Synthesis of (PEG₄₀-FMS)-2-hGH

(PEG₄₀-FMS)-2-hGH was prepared by the two-step procedure described inExample 16, as follows: To a stirred solution of human growth hormone(4.4 mg, 0.22 μmol) in phosphate buffer (1 ml, 0.1 M, pH 7.2),MAL-FMS-NHS (4 eq, 0.47 mg, 0.8 μmol) was added. The reaction wascarried out for 30 min at 25° C., and then dialyzed against H₂O (pH 6)at 4° C. for 24 h, to remove excess MAL-FMS-NHS. Solid PEG₄₀-SH (7 eq,65 mg, 1.5 mmol) was added, and the reaction proceeded for 2 h inphosphate buffer (1 ml, 0.1M, pH 7.2, containing 2 mM ascorbic acid).The thus obtained title compound was purified by RP-HPLC using a C4column, and characterized by SDS gel electrophoresis (12.5%) indicatingthe presence of (PEG₄₀-FMS)-2-hGH.

Example 25 Synthesis of PEG₄₀-FMS-hGH

MAL-FMS-NHS (280 μg) was added to a solution of hGH (4.5 mg, 0.225 mmol)in 0.5 ml PBS, pH=7.4. After 10 min, PEG₄₀-SH (10 mg) was added and thereaction mixture was stirred for 1 h and dialyzed overnight againstwater. The product, PEG₄₀-FMS-hGH, was purified by RP-HPLC.

Example 26 Synthesis of PEG₄₀-FMS-ANP

To a stirred solution of atrial natriuretic peptide (ANP) (0.32 mmol, 1mg) in phosphate buffer (1 ml, 0.1 M, pH 7.2), MAL-FMS-NHS (2 eq, 0.2mg, 0.7 μmol) was added. The reaction was carried out for 10 min at 25°C. Solid PEG₄₀-SH (2.2 eq, 30 mg, 0.7 mmol) was added, and the reactionproceeded for 2 h in phosphate buffer (1 ml, 0.1M, pH 7.2, containing 2mM ascorbic acid). The product was further purified by HPLC.

II. Biological Section

In this section, the biological activity of the PEG-Fmoc and PEG-FMSconjugates with gentamicin, peptides and proteins prepared in theexamples above, was tested.

Example 27 Inactive PEG-Fmoc-Gentamicin Conjugates Undergo Reactivation

For assaying the antibacterial potency of gentamicin and itsderivatives, a suspension of Escherichia coli (E. coli strainN-4156-W.T, 1% v/v in LB medium) was divided into plastic tubes (0.5 mlper tube) and incubated in a shaking water bath at 37° C., in either theabsence or the presence of increasing concentrations of gentamicin (0.02to 2 μM), and increasing concentrations of PEG_(5,000)-gentamicinderivatives (0.02-50 μM). E. Coli replication was evaluated by measuringthe absorbance at 600 nm. Incubation was terminated when O.D₆₀₀ nm inthe tubes containing no gentamicin reached a value of 0.6±0.1. Under ourassay conditions, native gentamicin inhibited half-maximally E. Colireplication at a concentration of 0.22±0.02 μM (0.1 μg/ml). APEG-gentamicin derivative showing an IC₅₀ value of 2.2±0.2 μM in thisassay is considered having 10% the antibacterial potency of nativegentamicin.

(PEG_(5,000)-Fmoc)₁-gentamicin and (PEG₅₀₀₀-FMoc)-2-gentamicin,containing one or two moles of PEG_(5,000)-Fmoc/mol gentamicin,respectively, were prepared as a model in order to assess thereversibility of the PEG-Fmoc moieties. Both derivatives (0.2 mM ofeach) were incubated in 0.1 M NaHCO₃ (pH 8.5) at 37° C., and aliquotswere withdrawn at different times and then analyzed for their potency toarrest E. coli replication (see method (xi) above). The IC₅₀ for eachaliquot was determined. Native gentamicin inhibited E. Coli replicationwith IC₅₀ value of 0.22±0.2 μM. (PEG_(5,000)-Fmoc)₂- and(PEG_(5,000)-Fmoc)₁-gentamicin had 0.01 and 2.1±0.1% of theantibacterial potency of gentamicin, respectively.

The results are shown in FIGS. 3A-3B. (PEG₅₀₀₀-Fmoc)-2-gentamicin showeda prolonged lag period (about 15 hours) prior to noticeable reactivation(FIG. 3A). A sharp elevation in antibacterial potency then occurred,reaching 50 or 100% of the native potency after 30±2 or 60±3 hours ofincubation, respectively (FIG. 3A). No lag period was observed uponincubation of (PEG_(5,000)-Fmoc)₁-gentamicin, Reactivation proceededcontinuously with half-maximal reactivation at 6±0.3 hours, and fullactivation (100%) after 30 hours of incubation (FIG. 3B).

Example 28 Biological Activity of PEG-Fmoc-Insulin Conjugates Materialsand Methods

(i) Materials. Recombinant human insulin was from Biotechnology General(Rehovot, Israel). D-[U-⁴C]Glucose (4-7 mCi/mol) was obtained from DuPont NEN (Boston, Mass., USA). Collagenase type I (134 U/mg) waspurchased from Worthington (Freehold, N.J., USA).

(ii) Rat adipocytes were prepared from fat pads of male Wistar rats(100-200 gr) by collagenase digestion as described (Rodbell, 1964). Thefat pads were cut into small pieces with scissors and suspended in 3 mlof KRB buffer containing NaCl, 110 mM; NaHCO₃, 25 mM; Kcl, 5 mM; KH2P0₄,1.2 mM; CaCl₂, 1.3 mM; MgSO₄, 1.3 mM; and 0.7% BSA (pH 7.4). Digestionwas performed with collagenase type I (1 mg/ml) in a 25 ml flexibleplastic bottle under an atmosphere of carbogen (95% O₂, 5% CO₂) for 40min at 37° C. with vigorous shaking. Five ml of buffer was then added,and the cells were passed through a mesh screen. The cells were thenallowed to stand for several minutes in a 15 ml plastic test tube atroom temperature, floating, and the buffer underneath was removed. Thisprocedure (suspension, floating, and removal of buffer underneath) wasrepeated three times.

(iii) Lipogenesis (incorporation of [U-¹⁴C] glucose into the lipids ofintact adipocytes). The incorporation of [U-¹⁴C] glucose into adiposetissue in rat adipocytes was performed as described (Moody et al.,1974). Adipocyte suspensions (3×10⁵ cells/ml) were divided into plasticvials (0.5 ml per vial) and incubated for 60 min at 37° C. under anatmosphere of carbogen with 0.2 mM [U-¹⁴C]glucose, in either the absenceor presence of insulin. Lipogenesis was terminated by addingtoluene-based scintillation fluid (1.0 ml per vial) and theradioactivity in extracted lipids was counted. In a typical experiment,insulin-stimulated lipogenesis was 4-5 fold higher than basal (basal:2000 cpm per 3×10⁵ cell/h; Vinsulin 8,000-10,000 cpm per 3×10⁵ cells/h).In this assay, insulin stimulates lipogenesis with ED₅₀ value=0.15±0.03ng/ml. An insulin analog exhibiting ED₅₀ value=15 ng/ml is considered tohave ˜1% of the biological potency of the native hormone.

(iv) Glucose-lowering potency of insulin and its derivatives wasdetermined in mice following administration under the conditionsspecified in each experiment. Blood samples were taken from the tailvein at different time points after administration, and blood glucoselevels were determined with glucose analyzer (Beckman Instrumen,Fullerton, Calif., USA) by the glucose oxidase method. The level ofglucose in normal healthy CD1-mice was 140-7 mg/dl (7.77 mM). Each groupconsisted of 4-5 mice. Data are presented as means±SEM.

28 (i). Progressive Modification of Amino Acid Moieties of Insulin withPEG₅₀₀₀-Fmoc-OSu

Human insulin was modified with increasing concentrations of Precursor Iand loss of biological potency as a function of PEG₅₀₀₀-Fmocincorporated into insulin was determined.

Insulin (17.24 nmoles in 0.2 ml 0.01 M NaHCO₃) reacted with increasingconcentrations of PEG₅₀₀₀-Fmoc-OSu at a molar excess over the protein asindicated in FIG. 4A for 2 hours at 25° C. The degree of derivatizationwas quantitated by determining the number of free amino groups thatremained unmodified and were available for reaction with TNBS.Theoretically, insulin can incorporate 3 moles of PEG₅₀₀₀-Fmoc on theamino side chains of Lys B29, PheB1 and GlyA1. As shown in FIG. 4A, uponreacting insulin with 0.6, 1.3, 1.9, 2.5 and 5.0 molar excess ofPEG₅₀₀₀-Fmoc-OSu, 0.3±0.03, 0.59±0.05, 1.1±0.1, 1.5±0.2 and 2.2=0.2moles of PEG_(5,000)-Fmoc were incorporated into insulin, respectively,indicating that two (of the three) amino side chains of insulin arereadily accessible for derivatization.

Aliquots containing 0.4, 0.7, 1.1, 1.5 and 2.2 moles PEG₅₀₀₀-Fmoccovalently attached per mole insulin, were assayed for their biologicalpotencies in a lipogenic assay in rat adipocytes. Under the assayconditions, human insulin stimulates lipogenesis, 4-6 times above basallevels with ED₅₀ value of 0.2±0.02 ng/ml. An insulin derivativeexhibiting ED₅₀ of 2.0±0.2 ng/ml in this assay is considered as having10% the lipogenic potency of native insulin. The results in FIG. 4B showthat the respective biological potencies were 87±4, 60±3, 8±1, 4±0.3 andless than 1% when 0.3, 0.6, 1.1, 1.5 and 2.2 mol PEG₅₀₀₀-Fmoc/molinsulin has been incorporated.

28(ii). PEG₅₀₀-Fmoc-Insulins Undergo Time-Dependent Reactivation

Insulin conjugates containing one and two moles of PEG₅₀₀₀-Fmoc/moleinsulin were incubated for different times at a concentration of 0.172μM in 0.1 M NaHCO₃-0.5% bovine serum albumin (BSA) and 1 mM NaN₃ (pH8.5) at 37° C. The results are shown in FIG. 5. At the indicated timepoints, aliquots were analyzed (in several concentrations for eachaliquot) for their lipogenic potencies in rat adipocytes. With(PEG₅₀₀₀-Fmoc)-2-insulin, no reactivation could be observed in the first10 hours of incubation. Lipogenic activity then increased, reaching 45±3and 90% of the native insulin potency at 30±2 and 80±4 hours,respectively.

With (PEG₅₀₀₀-Fmoc)₁-insulin, no such lag period was observed. Activitywas regenerated slowly but continuously yielding 16, 24, 30, 36 and 95%of the native potency following 2, 4, 10, 17 and 80 hours of incubation,respectively. Insulin containing about one mole of PEG₅₀₀₀-Fmoc/molinsulin (PEG₅₀₀₀-Fmoc₁-insulin) was therefore selected for furtherstudies. Mass-spectrum and analytic HPLC analyses revealed that thispreparation contains predominantly monomodified derivatives of insulin(MW=11,096 kDa), the remainder being unmodified insulin (about 5%,MW=5,813 kDa) and small quantities of bis (MW=16,587 kDa) and tris(MW=22,661 kDa) modified derivatives.

As shown in FIG. 4B, (PEG₅₀₀₀-Fmoc)₁-insulin has 7±1% the biologicalpotency of the native hormone prior to undergoing PEG-Fmoc hydrolysis.Based on these findings in vitro, (PEG₅₀₀₀-Fmoc)₁-insulin wasadministered in vivo at ten times higher concentrations than the nativehormone, in order to obtain the same glucose-lowering effect (seebelow).

28(iii). (PEG₅₀₀₀-Fmoc)₁-Insulin Facilitates Prolonged Glucose-LoweringAction In Vivo

Native insulin (Zn²⁺ free, 1.72 nmol/mouse) or (PEG₅₀₀₀-Fmoc)-1-insulin(17.2 nmoles/mouse) were administered subcutaneously to groups of mice(n=5 in each group), and the glucose lowering profiles were determined.The results are shown in FIG. 6. Native insulin reduced blood glucoselevel maximally at 30 min. Circulating glucose levels then returned tonormal values with t_(1/2)=1.8±0.2 hours. Following subcutaneousadministration of (PEG-Fmoc)₁-insulin, circulating glucose levels werelowered maximally at 4 hours, and were then maintained at the lowglucose level for an additional 4 hours, before returning to the normalvalues with a t_(1/2)=12±1 hours (FIG. 6).

28(iv) (PEG₅₀₀₀-Fmoc) 1-Insulin Manifests Prolonged Glucose LoweringAction Also after Intraperitoneal Administration

The prolonged action of subcutaneously administered(PEG₅₀₀₀-Fmoc)₁-insulin can be attributed, theoretically, to a slowerabsorption rate from the subcutaneous compartment to the circulation aswell as to the low fraction of the administered material exposed toreceptor-mediated degradation and hydrolysis of the inactive to theactive species. In order to differentiate between these factors, thesubcutaneous compartment was bypassed by administering native insulin(Zn²⁺-free, 0.345 nmoles/mouse, in 0.2 ml PBS buffer) or(PEG₅₀₀₀-Fmoc)₁-insulin (3.45 nmoles/mouse, in 0.2 ml PBS buffer)intraperitoneally. The glucose lowering capacities were then monitored.Blood glucose levels were determined at different time points.

The results are shown in FIG. 7. Following administration of insulin,circulating glucose levels fell maximally at 15 min and returned tonormal level with a t_(1/2) value of 1.0±0.1 hours. In the case ofintraperitoneally administered (PEG₅₀₀₀-Fmoc)₁-insulin, circulatingglucose level declined more gradually, reaching a maximal fall at 1.0hour. Levels were then gradually elevated showing a t_(1/2) value of3.4±0.1 hours, and returning to normal values only 6 hours afteradministration (FIG. 7).

Example 29 Biological Activity of PEG-FMS-Exendin Conjugates

Exendin-4 is an insulinotropic glucagon-like peptide-1 (GLP-1) agonistassociated with the β-pancreatic cells, elevates endogenous cAMP levels,enhances secretion of insulin, and lowers circulating glucose levels(Eng et al., 1992; Goke et al., 1993; Schepp et al., 1994; Fehmann etal., 1994). A profound pharmacological advantage of this GLP-1 agonistis that, following administration at any dosage, the circulating bloodglucose level (BGL) never falls below a threshold glucose level that, innon-diabetic healthy CD1-mice, is 74±7 mg/dl (Shechter et al., 2003).

According to the present invention, the conjugate PEG₄₀₀₀₀-FMS-exendin-4was prepared (Example 19). Exendin-4 contains one His No amino functionand two Lys Nε amino groups, enabling modification at these threepositions. Indeed, N-terminal amino acid sequencing revealed that,although the PEG₄₀₀₀₀-FMS-exendin-4 product eluted as a single peak onHPLC, it was actually a mixture containing primarily the Nα-modifiedhormone. However, in view of the pegylation reaction's reversibilityaccording to the present invention, regenerating the native peptide andprotein hormones in physiological environment, this point deserves onlyminor consideration.

Materials and Methods

(i) Materials. Exendin-4 and a non-lysine-containing syntheticirrelevant peptide of 27 amino acids (SEQ ID NO: 14) were synthesized bythe solid phase method using a multiple-peptide synthesizer, AMS 422(Abimed Analyser Technik GmbH, Langenfeld, Germany). An Fmoc-strategywas employed throughout the peptide-chain assembly.5,5-dithiobis(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH) andtrinitrobenzene sulfonic acid (TNBS) were purchased from Sigma ChemicalCo., (St. Louis, Mo., USA). All other materials used in this study wereof analytical grade.

PEG₄₀₀₀₀-FMS-exendin-4 and PEG₅₀₀₀-FMS-exendin-4 were prepared asdescribed in Examples 18 and 19, respectively.

For the preparation of PEG₄₀₀₀₀-exendin-4, exendin-4 (0.3 mg, 75 nmol)was dissolved in PBS (pH=7.5) and reacted with PEG₄₀₀₀₀-OSu (20 mg, 470nmol) for 3 h, filtered (×7) through Centricon (cut off=50 kDa) andcharacterized by MALDI-TOF mass spectrometry (found 48074 Da).

PEG₄₀₀₀₀-FMS-Peptide 27 was prepared as described forPEG₄₀₀₀₀-FMS-exendin-4.

A Centricon-50 ultrafiltration device for aqueous solutions waspurchased from Millipore S.A. (France).

Ultraviolet spectra were obtained by Beckman DU 7500 spectrophotometerin 1 cm path length UV cuvettes. Mass spectra were determined usingMALDI-TOF and ESMS techniques (Bruker-Reflex-Reflectron model, Germany,and VG-platform-II electrospray single quadrupole mass spectrometer,Micro Mass, U. K., respectively).

(ii) HPLC analyses were performed using a Spectra-Physics SP8800 liquidchromatography system equipped with an Applied Biosystems 757 variablewavelength absorbance detector, and a Spectra-SYSTEM P2000 liquidchromatography system equipped with a Spectra-SYSTEM AS100 auto-samplerand a Spectra-SYSTEM UV1000, all controlled by a ThermoQuestchromatography data system (ThermoQuest Inc., San Jose, Calif., USA).The column effluents were monitored by Uv absorbance at 220 nm.Analytical RP-HPLC was performed using a pre-packed Chromlith™Performance RP-118e (4.6×100 mm, Merck, Darmstadt, Germany). The columnwas eluted with a binary gradient of 10-100% solution B over 10 min witha flow rate of 3 ml/min (solution A was 0.1% TFA in H₂O and solution Bwas 0.1% TFA in acetonitrile:H₂O; 3:1, v:v). PEGylated compounds wereanalyzed using a RP-4 column (250×4 mm, 5 μm bead size, VYDAC, Hesperia,Calif., USA) with a binary gradient of 10-100% solution B in 50 min at aflow rate of 1 ml/min.

(iii) Preparative separations were performed with pre-packed VYDAC RP-18or RP-4 columns (250×22 mm; Hesperia, Calif.). The column was elutedwith 10-100% solution B over 60 min (12 ml/min).

(iv) Glucose-lowering assay. Three groups of CD1 mice (n=6 per group)were subcutaneously administered with saline, native exendin-4 (4μg/mouse) or PEG₄₀₀₀₀-FMS-exendin-4 (4 μg peptide/mouse). Circulatingglucose levels were measured with a glucose analyzer (BeckmanInstrument, Fullerton, Calif., USA) by the glucose oxidase method. Bloodsamples for the blood glucose analyses were taken from the tail veins.The level of glucose in normal healthy CD1-mice was 140±7 mg/dl (7.77mM).

29(i) PEG-FMS-Exendin-4 Releases Exendin-4 Upon Incubation

A solution of PEG₄₀-FMS-exendin-4 (0.25 mM, 1 ml/ml in terms ofexendin-4 in 0.1 M NaHCO₃, pH 8.5) was incubated at 37° C. At differenttime points, aliquots (50 μl) were withdrawn and analyzed for therelease of exendin-4 from the conjugate, using HPLC on a RP-4 column. Asshown in FIG. 8, exendin-4 was released from the conjugate[PEG₄₀-FMS]₂-exendin-4 in a slow, homogeneous fashion, with a t_(1/2) of16-2 h. Exendin-4 was fully released from the conjugate after 48 h ofincubation.

29(ii) The Hydrolysis Rates and Reaction Orders of PEG₄₀-FMS-Exendin-4,PEG₅₀₀-FMS-Exendin-4 and PEG₅₀₀-FMS-4-Nitro-Phenethylamine

The hydrolysis rates and reaction orders of the PEG-FMS conjugatesPEG₅₀₀₀-FMS-4-nitro-phenethylamine, PEG₅₀₀₀-FMS-exendin-4 andPEG₄O-FMS-exendin-4, and one MAL-FMS conjugate, N-(MAL-FMS)-Peptide 27,were evaluated at pH=8.5, 37° C. The structures and half-lives of theconjugates are presented in Table 3. PEG₅₀₀₀-FMS-4-nitro-phenetylaminewas prepared as described in Example 20. The conjugate MAL-FMS-Peptide27 was prepared by reaction of MAL-FMS-NHS with the non-relevant peptide27 (SEQ ID NO:14).

FIG. 9 shows the hydrolysis of PEG₅₀₀₀-FMS-exendin-4 (circles) and ofPEG₅₀₀₀-FMS-4-nitro-phenethylamine (squares) after incubation in PBS atpH 8.5, 37° C. Results in FIGS. 8 and 9 are expressed as percent of themaximal peak area of released exendin-4 and 4-nitrophenethylamine, as afunction of time.

The hydrolysis rate at pH 8.5 is equivalent to the hydrolysis rate inserum. The release of 4-nitro-phenethylamine and exendin-4 was monitoredby RP-HPLC and was determined to be a first order reaction (Table 3 andFIGS. 8, 9). As shown (FIGS. 8-9, Table 3), the peptides and proteinwere released from the conjugates in a slow homogenous fashion, witht_(1/2) of 9.4 (PEG₅₀₀₀-FMS-4-nitro-phenethylamine), 13.8(PEG₅₀₀₀-FMS-exendin-4), and 11.9 (PEG₄₀₀₀₀-FMS-exendin-4). Exendin-4was fully released from the PEG₄₀ conjugate after 48 h of incubation.

The conjugate MAL-FMS-Peptide 27 is TNBS negative as the sole α-aminomoiety of Peptide 27 was derivatized. A solution of MAL-FMS-Peptide 27(0.5 mM in 0.1 M NaHCO₃, pH 8.5) was incubated at 37° C. Aliquots (0.2ml) were withdrawn at different time points and analyzed for theappearance of the free α-amino group using TNBS. The FMS-MAL (N-ethylmaleimide) moiety was hydrolyzed in a slow and nearly homogeneousfashion from the α-amino moiety of the peptide, with a t_(1/2) value of8.4 h. Hydrolysis was complete after 32 hrs of incubation.

29(iii) PEG₄₀-FMS-Exendin-4 Facilitates Prolonged Glucose LoweringAction in Mice

FIGS. 10A-10B show the glucose-lowering profile of subcutaneouslyadministered native exendin-4 and of PEG₄₀-FMS-exendin-4, both at a doseof 10 μg (FIG. 10A) or 4 μg (FIG. 10B) peptide/mouse relative to asaline-treated group of mice.

FIG. 10A shows the glucose lowering profile of subcutaneouslyadministered native-exendin-4 and of PEG₄₀-FMS-exendin-4, both at a doseof 10 μg/mouse. With the native peptide, blood glucose levels declinedfrom 139±10 to 96±7 within 0.5 h reaching a maximal fall in 2-4 hoursafter administration (74 mg/dl). The return to initial glucose level wasthen proceeded with a t_(1/2) value of 14.21 h. Following thesubcutaneous administration of PEG₄₀-FMS-exendin-4, little decrease inblood glucose level is seen at 0.5 h after administration. Circulatingglucose level then fall gradually, with the lowest glucose concentrationreached at 6-8 hours after administration (80-90 mg/dl). Stable, lowcirculating glucose concentrations then maintained over nearly 50 hours,prior to the return to initial glucose levels with a t_(1/2) value of70±3 hrs.

FIG. 10B shows the glucose lowering profile of subcutaneouslyadministered native-exendin-4 and of PEG₄₀-FMS-exendin-4, both at a doseof 4 μg/mouse. With the native peptide, blood glucose levels declined by26-28% (from 140 mg/dl to 104-101 mg/dl), with the largest percentchange in blood glucose levels occurring 0.5-1 h after administration.Glucose concentrations then returned to their initial levels with at_(1/2) value of 3.7±0.3 h. Following the subcutaneous administration ofPEG₄₀-FMS-exendin-4, the decrease in blood glucose level took place at amore moderate rate. Circulating glucose reached its lowest concentration8-12 hours after administration (92 mg/dl, 33%). Stable, low circulatingglucose concentrations were then maintained for a further 12 hours.Return to initial glucose levels took place with a t_(1/2) of 30±2 h,being 7.5 times longer than that obtained by the same dose of the nativehormone.

Calculations based on the hydrolysis rates of FMS-exendin-4 (describedin U.S. patent application Ser. No. 10/408,262) andPEG₄₀₀₀₀-FMS-exendin-4 at 37° C. in normal sera and PBS (pH 8.5),respectively, revealed that ˜4% of exendin-4 is released from theconjugate each hour in vivo (Shechter et al., 2001). We furtherhypothesized that this release rate, if combined with prolongedmaintenance of the conjugate in the circulatory system, prior toexendin-4 hydrolysis, should yield a long-lasting glucose-loweringsignal in mice, as indeed is found following a single subcutaneousadministration of PEG₄₀₀₀₀-FMS-exendin-4 conjugate (FIG. 10B).Irreversibly conjugated PEG₄₀₀₀₀-exendin-4 has only ≦1% of the activityof native exendin-4 (IC₅₀=250±30 μmol/mouse versus IC₅₀=2.5±0.24μmol/mouse for the native exendin-4, not shown).

Example 30 Biological Activity of PEG₄₀-FMS-IFNα2

Type I interferons (IFNs) are proteins that initiate antiviral andantiproliferative responses. Interferons are clinically important, andseveral subtypes of IFNα2 were approved as drugs for the treatment ofhepatitis B and C as well as for cancers such as chronic myelogenousleukemia and hairy cell leukemia. Interferons regulate signals throughthe Janus tyrosine kinase (Jak/STAT proteins), and by reducingphosphorylation and activation of MEK1 and ERK1/2 through a Ras/Rafindependent pathways (Romerio et al., 2000). Human type I interferonsinduce differential cellular effects, but act through a common cellsurface receptor complex comprised of the two subunits, Ifnar1 andIfnar2. Human Ifnar2 binds all type I IFNs, but with a lower affinityand specificity than the Ifnar complex. Human Ifnar1 has a low intrinsicbinding affinity towards human IFNs, but modulates specificity andaffinity of other ligands of the Ifnar complex (Cutrone and Langer,2001).

IFNα2 may be administered intramuscularly, subcutaneously orintravenously, resulting in different pharmacokinetic profiles. In anymode, the administered cytokine is rapidly inactivated by body fluidsand tissues (O'Kelly et al., 1985), and cleared from the blood plasmaseveral hours following administration (Rostaing et al., 1998). Themajor routes of IFNα2 elimination from the circulatory system arethrough proteolysis, receptor mediated endocytosis and kidney filtration(Goodman and Gilman, 2001).

Prolonging the maintenance dose of IFNα2 in circulation is a desirableclinical task. A non-reversible, 12 kDa-PEG-IFNα2 conjugate, has beentherapeutically approved in 2001. It is administered once a week tohepatitis C patients, and facilitates a sustained anti-viral responserate of 24%, as opposed to a 12% response rate obtained by the nativecytokine (Schering-Plough Corporation, 2001 press release; Baker, 2003).However, while the covalent attachment of PEG chains to proteinsprolongs their lifetime in vivo, it often results in a dramaticreduction or even loss of biological and pharmacological activities(Fuertges and Abuchowski, 1990; Katre, 1993; Bailon and Berthold, 1998;Nucci et al., 1991; Delgado et al., 1992; Fung et al., 1997; Reddy,2000; Veronese, 2001). The pegylated formulation of IFNα2 currently inuse has 7% the activity of the native cytokine, calling for higher dosesto be administered (Bailon et al., 2001). Furthermore, PEG-IFN does notreadily penetrate all tissues: while 12 kDa PEG-IFNα2b is widelydistributed, 40 kDa-PEG-IFNα2a is restricted to the blood and theinterstitial fluid (Glue et al., 2000; Reddy et al., 2002). This majordrawback can be overcome by designing a PEG-IFNα2 conjugate capable ofgenerating native IFNα2 at a slow rate under physiological conditions.

These problems of the prior art can be overcome by the mono- andhis-PEG₄₀-FMS-IFN-α2 conjugates of the invention, in which IFN-α2 islinked to the PEG moiety through the FMS moiety that provides the slowlyhydrolysable bond. These novel reversibly pegylated-conjugates and theirprolonged anti-viral activity in vivo are discussed here in detail.

Materials and Methods

(i) Materials. Non-glycosylated human IFN-α2 was prepared as describedin in WO 02/36067 as previously described by Piehler and Schreiber(1999). Preparation of PEG₄₀-FMS-IFNα2 and (PEG₄₀-FMS)₂—IFNα2 isdescribed in Examples 21 and 22, respectively.

(ii) Receptor binding affinities were evaluated by BIAcore (SPRDetection) measurements. The BIAcore®3000 system, sensor chips CM5, HBS(10 mM Hepes, 3.4 mM EDTA, 150 mM NaCl, 0.05% surfactant P20, pH 7.4)and the amine coupling kit were from BIAcore (Sweden). Chipimmobilization by Ifnar2 and the BIAcore measurements were carried outaccording to Pichler and Schreiber (2001). In short, Ifnar2-EC wasimmobilized to the surface using the non-neutralizing anti-Ifnar2-EC(Ifnar2-extra cellular) mAb 46.10, followed by cross-linking with asecond mAb (117.7) (kindly provided by D. Novick, Weizmann Institute ofScience, Rehovot, Israel). The binding curves were evaluated with theBIAevaluation software (Biacore AB, Sweden) using a simple one-to-onekinetic model. Increase in RU (resonance unit) after specific binding tothe receptor corresponds to the amount of protein bound to the sensorsurface. To estimate the increase in RU resulting from the nonspecificeffect of the protein on the bulk refractive index, binding of theprotein to a control surface with no immobilized ligand was alsomeasured and subtracted. For the determination of the active interferonconcentration the equilibrium response was plotted against the estimatedinitial concentration. The data were fitted using KaleidaGraph (version3.0.4, Abelbeck Software) using the equation:

$y = \frac{1*10^{8}*m\; 1*m\; 2}{{1*10^{8}*m\; 1*m\; 0} + 1}$

whereby a K^(A) of 1×10⁸ was determined independently for IFNα2 bindingand fixed for all samples, m1=is Ru/Rmax (the percent of activeinterferon measured in the sample), m2 is Rmax, and m0 is the observedRu.

(iii) In vivo experiments were performed using male Wistar rats (150-170g). Rats were injected either subcutaneously or intravenously (0.2ml/rat).

(iv) Antiviral activity of IFN^(α)2 and its derivatives was determinedby the capacity of the cytokine to protect human amnion WISH cellsagainst Vesicular Stomatitis Virus (VSV)-induced cytopathic effects(Rubinstein et al., 1981).

(v) Simulations of experimental data were performed using Pro-KineticistII, a 2^(nd) Order Global Kinetic Analysis software (AppliedPhotophysics Ltd., England).

For the i.v. administration, the following model was considered:

Whereby, k₁=0.01 hr⁻¹ and k₂=0.65 hr⁻¹ (determined experimentally byantiviral assay).

For the s.c. administration, the following model was considered:

Whereby, k₁=0.02 hr⁻¹, k₂=0.01 hr⁻¹, and k₃=0.65 hr⁻¹.

k₁, which could not be determined directly, was estimated to be 0.02hr⁻¹ from the fit of the simulation to the experimental data.

30(i) PEG₄₀-FMS-IFNα2 has Modified Receptor-Binding Capacity

The binding capacity of the modified IFNα2 of the invention toward theimmobilized recombinant extracellular part of IFNα2 receptor (ifnar2-EC)was monitored under flow-through conditions by an optical probe calledreflectometric interference spectroscopy (RIFS). This method detectsbiomolecular interactions of ligands to transducer-bound proteins as ashift in the interference spectrum caused by change of the apparentoptical thickness of the transducer chip. A shift of 1 pm corresponds toapproximately 1 pg/mm² protein on the surface. The transducer surfacewas modified with a dextran layer and carboxylated by reaction withmolten glutaric anhydride (Sigma) at 75° for 2-8 h. On such surfaces,electrostatic pre-concentration and covalent immobilization of proteinswere carried out by standard BIAcore protocols. After this procedure,ifnar2-EC was immobilized into a carboxylated dextran layer. Allmeasurements were carried out in 50 mM Hepes (pH 7.4) containing 150 mMNaCl and 0.01% Triton X-100. A sample of 0.8 ml was injected for 80 swith a data acquisition rate of 1 Hz. Flow rates of 50 μl/s wereapplied. Under these conditions, the samples in the flow cell wereexchanged within one second, allowing the analysis of processes within 5seconds.

The results are summarized in Table 4. Monomodified and bis-modifiedconjugates of PEG₄₀-FMS-IFNα2 had 9±1 and 0.4±0.05% the receptor-bindingcapacity of IFNα2, respectively. These conjugated derivatives, however,underwent time-dependent reactivation upon incubation at 37° C. inphosphate-buffer pH 8.5 or in normal human serum (not shown).Reactivation proceeded with a t_(1/2) value of 9±1 and 24±3 hrs for themono and bis-pegylated derivatives, respectively reaching nearly fullreactivation following 50 hours of incubation (Table 4).

30(ii) PEG₄₀-FMS-IFN_(α)2 Releases Native-Active IFNα2 Upon Incubation,at a Rate Constant of 0.01 hr⁻¹

In the set of experiments summarized in FIGS. 11A-C, PEG₄₀-FMS-IFNα2 wasincubated in 0.1 M phosphate buffer in the presence or absence of 0.6%BSA and 2 mM sodium azide (NaN₃) (pH 8.5, 37° C.). At this pH value, therate of FMS hydrolysis from FMS-proteins is nearly identical to thatobtained in normal human serum, in vitro, or in the circulatory systemin vivo (Gershonov et al., 2000; Shechter et al., 2001 and 2002).Aliquots were drawn at different time points and analyzed for therelease of IFNα2 from the conjugate by SDS-PAGE (FIG. 11A) and byBIAcore, measuring the active concentration of Ifnar2 according to thelaw of mass action (FIG. 11B). The interferon-binding curve on theIfnar2 surface resembles that of a homogeneous population of nativeinterferon, suggesting that PEG₄₀-FMS-IFNα2 does not bind Ifnar2. Forthe SDS-PAGE analysis, the amounts of IFNα2 discharged were quantifiedrelative to an IFNα2 reference of known concentration and intensity. Inboth cases the discharge profiles are in good agreement. The 10% activeinterferon observed at time zero in the BIAcore profile is due to nativeinterferon present in the sample. The rate of discharge was determinedby fitting the quantity of active interferon to a single exponentialequation (FIG. 11C). Accordingly, IFNα2 is released from the conjugatewith a rate constant of 0.01 hr⁻¹ (FIG. 11). Upon 66 hours ofincubation, 50% of the IFNα2 in the conjugate is discharged and is fullyactive. From the extrapolation of the curve fit obtained, it is assumedthat nearly all of the interferon will eventually be released and regainfull activity. No BIAcore data was collected at very long time points askeeping proteins for weeks at 37° C. is not advisable.

30(iii) Subcutaneous Administration of PEG₄₀-FMS-IFN_(α)2 DramaticallyIncreased its Half-Life In Vivo

Next, we determined the half-life and activity of PEG₄₀-FMS-IFNα2 inrats. Human IFNα2 is not active in rats, albeit its concentration can bedetermined from the antiviral potency in rat serum by measuring theVSV-induced cytopathic effects in WISH cells (see Methods above,antiviral activity assay). Native IFNα2 or PEG₄₀-FMS-IFNα2 wereadministered subcutaneously to rats. Blood aliquots were drawn atvarious time points, and analyzed for their antiviral potency. Followingthe administration of the native unmodified IFNα2 (100 μg/rat),circulating antiviral activity declined with a t_(1/2) value of 1 hrreaching a level lower than 20 μM IFNα2, 12 h after administration (FIG.12).

The circulatory behavior of PEG₄₀-FMS-IFNα2 following subcutaneousadministration to rats (at doses of 12 μg/rat, 60 μg/rat, or 120 μg/rat)shows a clearly visible dose-dependent behavior (FIG. 12).Administration of 12 μg/rat of the conjugated IFNα2 yielded maintenancelevels of 70±10 μM IFNα2 which were maintained 56 hours followingadministration. When a 10-fold increase in PEG₄₀-FMS-IFNα2 wasadministered, IFNα2 was continuously present in the serum 56 hours at amolar concentration of 450 μM. Administration of 60 μg/rat of theconjugate resulted in interferon levels of 225 μM at 56 hours, and 25 μMat 200 hours. Native IFNα2 present in the administered sample(approximately 10% as determined by BIAcore) contributed to theinitially high levels of IFNα2 observed in the rats' serum. These valuesdisplay a clearance curve similar to that of native IFNα2. The remaining90% of the IFNα2 were slowly discharged from the conjugate.

30(iv) Intravenous Administration of PEG₄₀-FMS-IFNα2 to Rats

To eliminate the contribution of the subcutaneous exchange, both theconjugate and the native cytokine were administered to rats (30 μg/rat)intravenously. For native interferon, the same half-life was measured,indicating that it readily penetrates the circulatory system followingsubcutaneous administration (FIG. 13). For PEG₄₀-FMS-IFNα2, antiviralactivity was still detected 150 hours following intravenousadministration, demonstrating the prolonged effects of the pegylatedcytokine. Discharged IFNα2 level of 10 μM still remained 150 hours postadministration, while native IFNα2 was eliminated within 30 hourspost-administration. It should be noted that the large shoulder observedfollowing subcutaneous administration of the conjugate (FIG. 12) is notobserved when PEG₄₀-FMS-IFNα2 was administered intravenously (FIG. 13).

30(v) Simulations of Experimental Data

Using the rate constants obtained from both BIAcore data (k=0.01 hr⁻¹for the discharge of IFNα2 from the PEG conjugate) and the antiviralactivity assay of native IFNα2 (k=0.65 hr⁻¹ for the elimination ofinterferon), both the subcutaneous and intravenous administration modesof PEG₄₀-FMS-IFNα2 to rats were simulated (FIGS. 14A and 14B,respectively). In both cases, the simulated data and the experimentalresults were in good agreement. As became evident from the simulateddata, the passage of the conjugate, but not of the native interferon,from the subcutaneous volume to the bloodstream, proceeds at a slow rateand is in the order of the discharge of interferon from the conjugate.This explains the shoulder observed between 10-70 hours in the activeprotein concentration. As expected, this shoulder is not found whenPEG₄₀-FMS-IFNα2 is administered intravenously.

DISCUSSION

As mentioned before, the pegylating technology applied to therapeuticproteins often leads to a drastic loss in the biological and thepharmacological potencies of the conjugates. In principle, thisdeficiency can be overcome by introducing the PEG chains via a chemicalbond that is either sensitive to hydrolysis or can be cleavedenzymatically by serum proteases or esterases. A prerequisite conditionfor efficient pegylation is that the hydrolysis of the PEG chains fromthe conjugate is to take place at a slow rate, and in a homogenousfashion in vivo.

Two basic irreversible PEG-IFNα2 conjugates are in therapeutic use atpresent. The first, a 12 kDa-PEG-IFNα2, which satisfactorily permeatesinto tissues. This preparation, however, is a relatively short-lived invivo, since its low molecular mass (calculated mass=32 kDa) isinsufficient to markedly arrest kidney filtration. The secondformulation, a 40 kDa-PEG-IFNα2, is an extremely long-lived species invivo. This conjugate, however, has poor permeability into tissues.Following administration, the conjugate is distributed only in the bloodand in the intestinal fluids (Bailon et al., 2001). We have, therefore,anticipated that the two prerequisite features for an optimal PEG-IFNα2conjugate, namely, a prolonged maintenance in vivo combined with freeaccess to peripheral tissues, can be obtained by linking a slowlyhydrolyzable PEG₄₀-chain to IFNα2.

We have previously found that upon linkage of FMS to proteins, theFMS-protein conjugate undergoes hydrolysis at physiological conditionswith a desirable pharmacokinetic pattern (Gershonov et al., 2000;Shechter et al., 2001, 2002, 2003). The rate of FMS hydrolysis isdictated exclusively by the pH and the nucleophilicity of the serum,both of which are maintained in mammals under strict homeostasis(Shechter et al., 2001). We therefore based our new development on theFMS principle. In neutral, aqueous solutions, FMS moieties undergo slow,spontaneous hydrolysis, resulting in the regeneration of the nativeproteins (Shechter et al., 2001). For this purpose, NHS-FMS-MAL wassynthesized, enabling us to link sulfhydryl-containing PEG chains to theamino groups of peptides and proteins via the hydrolyzable FMS function.The working hypothesis was that an inactive PEG-interferon conjugatecould regenerate the native protein in its active form in a continuousfashion over a long period of time. The principal monomodifiedPEG₄₀-FMS-IFNα2 conjugate obtained (Table 2) is devoid of the cytokinebinding potency and can therefore be referred to as a ‘prodrug’. Uponincubation, the native cytokine is released by hydrolysis, and thebinding potency of IFNα2 to Ifnar2 is regenerated with a rate constantof 0.01 hr⁻¹.

A single subcutaneous administration of PEG₄₀-FMS-IFNα2 significantlyprolonged the levels of IFNα2 in the serum of rats. While IFNα2 wasshort-lived in vivo, having a half-life of ˜1 hr, the PEG₄₀-FMS-IFNα2conjugate exerted its antiviral activity over a period of 200 hours.Furthermore, there is a dose-dependent ratio between the quantityadministered and the active interferon levels over a prolonged durationin vivo. This observation is beneficial for optimization of dosingregimens in future clinical use.

It should be noted that the IFNα2 molecule contains 13 amino functionstheoretically available for PEG₄₀-FMS attachment. The exact site ofpegylation was not determined. In view however, of its reversibility andthe generation of the native protein, it seems that this point deservesa rather minor consideration.

In summary, following the new conceptual approach forreversible-pegylation of the invention, whereby a pharmacologically‘silent’ conjugate is ‘trapped’ in the circulatory system and releasesthe parent protein with a desirable pharmacokinetic profile, we havesucceeded in combining prolonged maintenance of IFNα2 in vivo with therelease of active-native IFNα2 to ensure access to peripheral tissues.

Example 31 Biological Activity of the PEG₄₀-FMS-PYY₃₋₃₆ Conjugate

The hypothalamic family of neuropeptide Y (NPY) receptors plays a majorrole in regulating satiety and food intake (Schwartz, 2000). Theputative inhibitory Y2 pre-synaptic receptor (Y2R) is expressed in thearcuate nucleus, which is accessible to local and peripheral agonists ofthe NPY family (Broberger et al., 1997; Kalra et al., 1999). One suchY2R agonist is peptide YY₃₋₃₆ (PYY₃₋₃₆), which is released from thegastrointestinal tract post-prandially in proportion to the caloriccontent of a meal (Pedersen-Bjergaard et al., 1996; Adrian et al., 1985;Grandt et al., 1994). Recently, it was demonstrated that peripheraladministration of PYY₃₋₃₆ inhibits food intake in humans, mice and ratsand reduces weight gain in rats [Pittner et al., 2002; Batterham et al.,2002, 2003). Thus, infusion of PYY₃₋₃₆ to reach the normal post-prandialcirculatory concentrations of this peptide lead to a peak in serumPYY₃₋₃₆ within 15 min, followed by a rapid decline to normal levelswithin 30 min. Despite this rapid clearance, administration of PYY₃₋₃₆to fasting individuals decreases their appetite and reduces food intakeby 33% within a 12 h period following PYY₃₋₃₆ administration.Furthermore, no compensatory food intake occurs over the next 12 h(Batterham et al., 2002). Therefore, PYY₃₋₃₆ may find a clinical use intreatment of obesity and its associated disorders, including type IIdiabetes mellitus and cardiovascular diseases (Schwartz and Morton,2002).

The short circulatory half-life of PYY₃₋₃₆ and its potential inmanagement of obesity prompted us to develop a longer-acting form ofPYY₃₋₃₆. Indeed, in our hands the satiety induced by PYY₃₋₃₆ in micelasted for only 2-4 h following subcutaneous (sc) injection. Asmentioned before, covalent attachment of PEG to proteins and peptidesprolong their half-life in vivo but often leads to a drastic loss oftheir biological or pharmacological activity. We found that PYY₃₋₃₆,pegylated using standard chemistry, i.e. through formation of a stablebond, indeed lost its biological activity. We have then prepared andtest here the biological activity of the PEG₄₀-FMS— PYY₃₋₃₆ pro-drug ofthe invention.

Materials and Methods

(i) Animals. C57BL/6J male mice (Harlan Labs) aged 9±1 week (20-30 gbody weight) were used. Mice were kept under controlled temperature(21-23° C.) and light conditions (light on 6:00-18:00) at the WeizmannInstitute of Science (Rehovot, Israel) animal facility. The mice wereacclimated for at least one week prior to the initiation of the study.Mice had free access to drinking water at all times during theexperiments. All experimental protocols were in accordance with theIsraeli regulations of laboratory animal welfare and were approved bythe Institutional Internal Committee for Animal Welfare.

(ii) Reagents. Peptide YY₃₋₃₆ was synthesized by the solid phase method,using a multiple-peptide synthesizer AMS 422 (Abimed Analyser TechnikGmbH, Langenfeld, Germany). The resulting peptide was HPLC-purified andcharacterized by MALDI-TOF mass spectroscopy (MS) and N-terminal proteinmicro-sequencing. All other reagents were of analytical grade and werepurchased from Sigma Chemical Co. (Ness Ziona, Israel).

(iii) Analytical Procedures. Mass spectra were determined usingMALDI-TOF and Electro Spray (ES-MS) techniques (Bruker-Reflex-Reflectronmodel, Germany, and VG-platform-II electrospray single quadropole massspectrometer, Micro Mass, U. K., respectively). PYY₃₋₃₆,PEG₄₀-FMS-PYY₃₋₃₆ and PEG40 were resolved by analytical HPLC (LichrosorbRP-4 column, 4×250 mm, Merck). Buffer

A was 0.1% aq. trifluoroacetic acid (TFA) and Buffer B was 0.1% TFA in75% aq. Acetonitrile. A gradient of 10-100% Buffer B was used over 50min. at a flow rate of 1 ml/min. The retention times (Rt) of PYY₃₋₃₆,PEG₄₀-FMS-PYY₃₋₃₆ and PEG₄₀-9-sulfo-fulvene were 21.53, 39.03 and 44.387min., respectively. Amino acid analyses were performed following 6N HClhydrolysis at 110° C. for 24 h using a Dionex Automatic amino acidanalyzer HP1090 (Palo Alto, Calif., USA). N-terminal sequence analyseswere performed with a Model 491A Procise Protein sequencer (AppliedBiosystems, Foster City, Calif., USA).

(iv) Synthetic Procedures:

PEG₄₀-FMS-PYY₃₋₃₆ was prepared as in Example 23.

PEG₄₀-FMS-Glycine ethyl ester (PEG₄₀-FMS-GEE). To a stirred solution ofPEG₄₀-SH (0.25 mM in 0.1M phosphate buffer pH 7.2-10 mM Na ascorbate),292 μg MAL-FMS-NHS was added (two-fold molar excess over PEG₄₀-SH).After 7 min, 0.2 ml from a solution of 0.5M glycine ethyl ester wasadded. The reaction was carried out for 1 h, and the mixture was thendialyzed overnight against water. The resulting PEG₄₀-FMS-glycine ethylester was characterized and quantitated by its absorbance at 280nM/ε₂₈₀=21,200) and by the amount of glycine in the preparationfollowing acid hydrolysis of a 20 μl aliquot and amino acid analysis.

PEG₄₀-PYY₃₋₃₆. Irreversibly pegylated PYY₃₋₃₆ was prepared by reactingPYY₃₋₃₆ (1 mg/ml in 0.1 M phosphate buffer pH 7.2) with four equivalentsof solid PEG₄₀-OSu, (43 mg). The reaction was carried out for 1 h andthe mixture was then dialyzed overnight against water. The conjugatethus obtained contains one PEG₄₀ residue per PYY₃₋₃₆, as determined byMALDI-TOF MS. (PEG₄₀-OSu, 43,626 D; PYY₃₋₃₆, 4,047 D; conjugate, 47,712D).

(v) Food intake measurements. Groups of 10 mice were deprived of foodfor 24 h and then given excess pre-weighed standard chow for a period of2 h. Drinking water was provided at all times. The mice received scinjections of either saline (0.1 ml/mouse), native PYY₃₋₃₆ or PYY₃₋₃₆derivatives at the indicated doses and times within 24 h prior to thestart of the 2 h re-feeding period. A minimum interval of 1 h wasintroduced between injection and re-feeding to avoid a stress-dependentdecrease in food intake. The amount of food consumed per group wasdetermined at the end of the feeding period. Remaining chow was weighedafter the 2 h re-feeding period and the cumulative food intake per 10mice was calculated. Values of food intake in replicate experiments werenormalized according to the saline controls. Results are expressed asfood intake per 10 mice±SD from 2-5 replicate experiments.

(vi) Statistical analysis. The significance of differences in foodconsumption was determined by the Student's t-test, using the totalweight of food consumed by each group of 10 mice as a single value. Asaline-injected group of 10 mice was included in each experiment as acontrol. A two-tailed, paired t-test was performed between the controland treated groups.

31(i) The effect of PYY₃₋₃₆ on Food Intake in Mice

To evaluate the duration and magnitude of the effect of PYY₃₋₃₆ and itsderivatives on food intake, we employed the mouse re-feeding model of 24h starvation followed by a re-feeding period of 2 h. Initially werepeated previous studies, where PYY₃₋₃₆ was administered immediatelyprior to the re-feeding period. However, we found that the stress ofmere handling the mice had a profound and inconsistent effect on foodintake, thereby reducing the difference between saline and PYY₃₋₃₆. Wethen injected the mice sc 1 h prior to start of the re-feeding periodand obtained a much more consistent difference between PYY₃₋₃₆ andsaline. FIG. 15 shows that PYY₃₋₃₆ inhibited food intake in adose-dependent manner. Mice receiving saline or PYY₃₋₃₆ at a dose of 5nmol/mouse consumed 10.7±1.26 and 5.26±1.47 g chow per 10 mice,respectively (P<0.001, N=5). These figures correspond to a 50% decreasein food intake by the PYY₃₋₃₆ groups, as compared with the controlsaline groups. Inhibition was statistically significant at all dosesused, including the lowest dose of 0.2 nmol/mouse (0.8 μg/mouse, P>0.05,N=3). The dose-response corresponded to a half maximal effect of PYY₃₋₃₆at about 0.5 nmol/mouse (FIG. 15).

We then determined the duration of the satiety induced by scadministration of PYY₃₋₃₆ (5 nmol/mouse) at different times prior to there-feeding period. The satiety induced by PYY₃₋₃₆ was maximal when given1 h prior to re-feeding and rapidly decreased when PYY₃₋₃₆ wasadministered at earlier times. The half-life of its biological responsewas about 3 h and no effect was seen with 5 nmol PYY₃₋₃₆ whenadministered nearly 10 h prior to the re-feeding period (FIG. 16).

Intraperitoneal (rather than subcutaneous) administration of PYY₃₋₃₆ tomice has induced a considerably shorter-lived satiety effect. Forexample, an ip dose of 5 nmol PYY₃₋₃₆/Mouse was fully effective whenadministered 30 min before re-feeding, but had no effect whenadministered 2 h before meal (not shown).

We then attempted to extend the biological activity of PYY₃₋₃₆ byconventional pegylation. Thus, mono-pegylated PYY₃₋₃₆ was prepared (seeMaterials and Methods above) and tested for its biological activity bysc administration (5 nmol/mouse) 1 h prior to the re-feeding period. Nosignificant reduction in food intake was obtained as compared with thesaline control, indicating that conventional pegylation, which resultsin a stable pegylated peptide, abolished the biological activity ofPYY₃₋₃₆ (FIG. 17).

We then tested if the inactivation of PYY₃₋₃₆ was due to the bulkinessof the PEG group or due to blocking of the amino residues of PYY₃₋₃₆.Acetylation of the two amino groups of PYY₃₋₃₆ largely abolished theeffect of PYY₃₋₃₆ in inducing satiety in vivo. Thus, mice injected with20 μg Nα—Nε-diacetyl-PYY₃₋₃₆ per mouse ate 10.54±0.08 g chow per 10mice, as compared with 10.6±1.26 g in the saline group and 5.26±1.47 gin the PYY₃₋₃₆ group. Hence, even attachment of a small acetyl group wassufficient to disrupt the biological activity of PYY₃₋₃₆.

31(ii) Preparation and Characterization of PEG₄₀-FMS-PYY₃₋₃₆

The lack of biological activity following pegylation or even acetylationof PYY₃₋₃₆ prompted us to prepare the PEG₄₀-FMS-PYY₃₋₃₆ conjugatedescribed in Example 23 hereinabove. This product was analyzed bymass-spectrometry to determine the ratio of PEG₄₀ to PYY₃₋₃₆ byMALDI-TOF MS analyses. PYY₃₋₃₆ exhibited a molecular mass of 4047.51 D(calc.=4049.6 D). PEG₄₀-SH had an average mass of 43,626 D, and theconjugate yielded a major peak corresponding to a molecular mass of47,712.6 D (not shown). The calculated mass of the 1:1 conjugate ofPEG₄₀-SH and MAL-FMS-PYY₃₋₃₆ is 48,087 D. Thus the main productcorresponds to such a 1:1 conjugate.

31(iii) PEG₄₀-FMS is Linked to the α Amino Group of PYY₃₋₃₆

Peptide YY₃₋₃₆ contains the N-terminal α-amino group of isoleucine andone ε-amino group of lysine at position 2, both of which are potentiallyavailable for acylation by MAL-FMS-OSu. To determine the site(s) ofacylation, PEG₄₀-FMS-PYY₃₋₃₆ was reacted with a 500 molar excess ofacetic anhydride at pH 7.0 for 1 h, followed by dialysis against waterovernight. The PEG₄₀-FMS group was then removed by incubating theacetylated PEG₄₀-FMS-PYY₃₋₃₆ for 4 days at pH 8.5. The product was thensubjected to N-terminal protein sequence analysis. As shown in FIG. 18,cycles 1, 2 and 3 yielded the expected amounts of isoleucine, ε-acetyllysine and proline, respectively, No free lysine was found in cycle 2.Thus, the conjugate consists of PEG₄₀-FMS and PYY₃₋₃₆ at a 1:1 ratio.Furthermore, based on the sequencing yields, the PEG₄₀-FMS group isprimarily linked to the N-terminal α-amino group of PYY₃₋₃₆.Nevertheless, this analysis could not entirely exclude the presence of aPYY₃₋₃₆ mono-substituted at its E-lysine side chain. However, in view ofthe complete regeneration of the native peptide, this point is of arather minor significance.

31(iv) PEG₄₀-FMS-PYY₃₋₃₆ Hydrolyses to Yield PYY₃₋₃₆ Under PhysiologicalConditions

To evaluate the rate of PYY₃₋₃₆ release from PEG₄₀-FMS-PYY₃₋₃₆, weincubated the conjugate in phosphate buffer (pH 8.5, 0.1 M, 37° C.).Aliquots were withdrawn at different times and subjected to analyticalHPLC, using eluting conditions, which resolve PEG₄₀-FMS-PYY₃₋₃₆ fromPYY₃₋₃₆. At pH 8.5, the rate of FMS hydrolysis from FMS-peptides orproteins is nearly identical to that obtained in normal human serum(Shechter et al., 2001). As shown in FIG. 19, PYY₃₋₃₆ was released fromthe conjugate in a slow, nearly homogenous fashion with a half-life of5.3 h. After 40 h, the cumulative amount of free PYY₃₋₃₆ reached 79% ofthe input PEG₄₀-FMS-PYY₃₋₃₆.

31(v) Hydrolysis of PEG₄₀-FMS-PYY₃₋₃₆ in Normal Mouse Serum

We found that PYY₃₋₃₆ undergoes rapid proteolysis in normal mouse serumat 37° C. with a half-life of 3±0.7 min. (FIG. 20, insert). Therefore,the rate of PEG₄₀-FMS-PYY₃₋₃₆ hydrolysis was evaluated by the amount of2-PEG₄₀-9-sulfo-fluorene released during hydrolysis. PEG₄₀-FMS-PYY₃₋₃₆was incubated in normal mouse serum at 37° C. Aliquots were withdrawn atdifferent times, ethanol (3 volumes) was added to precipitate serumproteins, and following centrifugation the amount of2-PEG₄₀-9-sulfo-fluorene was measured in the supernatants by HPLC. Asshown in FIG. 20, hydrolysis of PEG₄₀-FMS-PYY₃₋₃₆ in normal mouse serumat 37° C. proceeded in a nearly homogenous fashion with a half-life of7.0±0.3 h.

31(vi) PEG₄₀-FMS-PYY₃₋₃₆ Induces a Prolonged Satiety in Mice

We then determined the duration of the effects of PEG₄₀-FMS-PYY₃₋₃₆ (5nmol/mouse) on food intake. The half-life of the biological response ofPYY₃₋₃₆ was about 3 h (FIG. 16). By comparison, the biological activityof PEG₄₀-FMS-PYY₃₋₃₆ was much more persistent (FIG. 21). Mice givenPEG₄₀-FMS-PYY₃₋₃₆ 18 h prior to re-feeding are 4.7±0.14 g chow per 10mice, a value representing a 52% reduction in food intake as comparedwith the control (saline) group (P>0.05). A statistically significantreduction of 27% in food intake was also seen in mice givenPEG₄₀-FMS-PYY₃₋₃₆ 24 h prior to re-feeding (P<0.05; FIG. 21). Thus, thehalf-life of the satiety effect exerted by PEG₄₀-FMS-PYY₃₋₃₆ was about24 h, namely, 8-fold longer than that of unmodified PYY₃₋₃₆. In acontrol study, sc administration of PEG₄₀-FMS-Glycine ethyl ester (5nmoles/mouse) 15 h before refeeding had no effect whatsoever on foodintake as compared with saline (FIG. 21, right columns).

Example 32 Biological Activity of (PEG₄₀-FMS)-2-hGH and PE G₄₀-FMS-hGH

Human growth hormone (hGH) is an essential pituitary hormone whichregulates growth and development of peripheral tissues. HGH is anFDA-approved drug that is widely in use for replacement therapy ingrowth hormone deficient children. As valid for other non-glycosylatedprotein drugs of molecular mass lower than 50(kDa, hGH is clearedrapidly from the circulatory system having a t_(1/2) value of 20-30 minin humans. Clearance takes place primarily via the kidney. The covalentattachment of PEG chains to hGH can substantially decrease clearance byglomerular filtration via the kidneys, and therefore elongate life-timein vivo. Several PEG chains must be introduced to hGH on order toappreciably elongate its life-time in vivo. Pegylation often results ina drastic decrease in the biological or pharmacological potencies ofpeptides and proteins. This is especially valid for hGH owing to therelatively large surface area of site 1 and site 2 through which thehormone binds the first receptor and then the second receptor to form ahomodimeric receptor complex that initiates signaling.

The covalent attachment of PEG chains to hGH in a nonreversible fashionhowever, leads to a drastic loss in the biological potency of thishormone. For instance, Clark et al., 1996, have linked two to sevenPEG₅₀₀₀ chains to the amino side chain moieties of hGH. The linkage oftwo PEG₅₀₀₀/hGH only already led to 90% loss in the biological potencyof hGH. About five such PEG₅₀₀₀ chains had to be linked to the proteinfor increasing circulating half-life substantially. Such (PEG₅₀₀₀)₅ hGHconjugates however had less than 0.1% the biological potency of hGH.

Two PEG-FMS-hGH conjugates have been prepared according to the invention(Examples 24-25). In fact, hGH contains several conjugatable aminofunctions. However, in view of the pegylation reaction's reversibilityaccording to the present invention and regeneration of the nativepeptide and protein hormones in physiological condition, this pointdeserves only minor consideration.

32(i) Receptor-Binding Capacity of (PEG₄₀-FMS)₂-hGH and PEG₄₀-FMS-hGH

The hGH displacement assay was performed as previously described(Tsushima et al., 1980). (PEG₄₀-FMS)-2-hGH, prepared by linking twoPEG₄₀-FMS moieties to hGH, has an effective molecular weight of about120 kDa and exhibits 9±2% the receptor binding potency of the nativehGH. Receptor binding capacity is regenerated upon incubation at 37° C.in normal rat serum or in 0.1M phosphate buffer (pH 8.5) at 37° C. witha t_(1/2) value of 20 hrs, reaching 70-80% the native-receptor bindingcapacity following 50 hours of incubation (not shown).

32(ii) The Hydrolysis Rate and Reaction Order of PEG₄₀-FMS-hGH

The hydrolysis rate and reaction order of PEG₄₀₀₀₀-FMS-hGH wereevaluated at pH=8.5, 37° C. The release of hGH was monitored by RP-HPLCand was determined to be a first order reaction (Table 3, FIG. 22). Invitro, PEG₄₀₀₀₀-FMS-hGH exhibits no receptor binding affinity. Uponincubation, recognition of the released hGH by the native hGH receptorwas preserved (FIG. 22). Human growth hormone was released from theconjugate in a slow homogenous fashion, with t_(1/2) of 11.8 h (Table 3,FIG. 23).

Example 33 PEG₄₀-FMS-ANP Undergoes Time-Dependent Hydrolysis atPhysiological Conditions

Atrial natriuretic peptide (ANP) is a 28-mer amino acid non-lysinecontaining peptide which exerts natriuretic, diuretic and vasorelaxantactions, It plays an important role in the body's blood volume and bloodpressure homeostasis. ANP has a very high affinity for its receptorsites (10 pm), and is therefore cleared very rapidly (t_(1/2)˜0.5 min)by receptor and protease-mediated events. ANP is therefore not suitableas a drug for subcutaneous administration for regulation of bloodpressure homeostasis in humans. The covalent attachment of a PEG₄₀-chainto the α-amino moiety of ANP generates a conjugate which is fully devoidof receptor binding affinity (data not shown).

PEG₄₀-FMS-ANP, prepared by linking PEG₄₀-FMS to the α-amino side chainof ANP, is TNBS negative. Upon incubation at pH 8.5 and 37° C., thisconjugate undergoes spontaneous hydrolysis with a t_(1/2) value of 23±2hrs. generating the parent ANP following 50 hours of incubation (Table5). Preliminary measurements in rats in vivo revealed thatSC-administered PEG₄₀-FMS-ANP has a 60-fold increase in serum half-life,in comparison to native ANP. The conjugate is fully protected fromreceptor-mediated degradation prior to the fall-off of the PEG₄₀-FMSchain from ANP by hydrolysis.

Example 34 An Albumin-Insulin Conjugate Releases Insulin Slowly UnderPhysiological Conditions

The covalent linkage of peptides or protein drugs to human serum albumin(HSA) greatly prolongs their lifetime in vivo, but is pharmacologicallyirrelevant when irreversibly inactivates them. We retain drugbioactivity by synthesizing a heterobifunctional reagent (Mal-Fmoc-OSu:9-hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene-N-hydroxysuccinimide)that generates HSA-Fmoc-insulin on covalent conjugation to insulin'samino group and HSA's cys-34 side chains. HSA-Fmoc-insulin iswater-soluble and, upon incubation in aqueous buffers reflecting normalhuman serum conditions, slowly, spontaneously and homogeneouslyhydrolyzes to release unmodified insulin with a t_(1/2) of 25±2 hrs. Asingle subcutaneous or intraperitoneal administration ofHSA-Fmoc-insulin to diabetic rodents lowers circulating glucose levelsfor about 4 times longer than an equipotent dose of Zn²⁺-free insulin.Following subcutaneous administration, onset of the glucose-loweringeffect is delayed 0.5-1 hr and persists for 12 hrs. Thus, we present aprototype insulin formulation possessing three desirable parameters:high aqueous solubility, delayed action following subcutaneousadministration and prolonged therapeutic effect.

Most polypeptide drugs, in particular nonglycosylated proteins ofmolecular mass less than 50 kDa, are short-lived species in vivo, havinga circulatory half-life of 5-20 min. The short lifetimes of proteins invivo are attributed to several mechanisms, including glomerularfiltration in the kidney and proteolysis at several levels. Insulin isdegraded primarily in the liver, through a mechanism defined asreceptor-mediated endocytosis. This mechanism is an efficient route forterminating the action of the hormone after the levels of glucose andother nutrients have been normalized. Chemically modified insulins withnegligible receptor binding affinities are therefore longer-livedspecies within the circulation, however they are biologicallyineffective.

IDDM patients receive multiple daily subcutaneous administrations of‘rapid’ and long-acting insulins. Prolonged long-acting insulinpreparations are needed to supply low basal levels of circulatinginsulin between meals and overnight, this being a physiologicalrequirement for reducing triglyceride breakdown and suppressing hepaticglucose output under ‘resting’ conditions.

Most long-acting insulins currently in use are suspensions of crystals,produced either by Zn²⁺ ions, or by the addition of basic protamine.Such injected suspensions have decreased rates of absorption from thesubcutaneous compartment to the circulatory system. Slow dissolution atthe site of injection brings about the protracted effect.

In recent years, major efforts have been devoted to prolonging theaction of insulins that are soluble in aqueous buffered media. In onesuch innovative approach, two arginine moieties were covalently linkedto insulin, to raise the isoelectric point of the hormone. Thederivative thus obtained is formulated in a soluble form in a slightlyacidic media, and is crystallized and precipitated immediately afterinjection, at the physiological, neutral pH of the subcutaneous space.

Albumin is long-lived in vivo. Similarly, drugs and endogenoussubstances that associate tightly with albumin have lower clearancerates than do the unbound substances, and exhibit prolonged life-timeprofiles in vivo (Taylor and Granger, 1984). Long chain-free fatty acidsbind tightly to albumin (Ka=10⁸M⁻¹ (Carter and Ho, 1994)), a fact thatprovided the impetus for preparation of soluble fatty acid acylatedinsulins that could bind to serum albumin. However, the affinity of theinsulin conjugates for albumin (Ka˜10⁵M⁻¹) was considerably less thanthat of the unbound long chain-free fatty acids and the conjugatesexhibited only moderately prolonged actions in vivo (Kurtzhals et al.,1995, 1996, 1997).

A series of HSA-peptide conjugates have been recently prepared throughformation of a stable covalent bond between maleimido-derivatives ofbiologically active peptides (e.g. ANP (Leger et al., 2003), dynorphine(Holmes et al., 2000), or the Kringle 5 region of plasminogen (Leger etal., 2004)) and the mercapto moiety of HSA's Cys-34. This was undertakenwith the aim of increasing the peptide's half-life in circulation byreducing elimination through the kidney and concomitantly protecting itfrom proteolysis by plasma enzymes. The activities of the HSA-boundpeptides, though significantly reduced as compared to the parentpeptides, were found to be rather substantial. Moreover, the conjugatesproved to be long-acting species.

Therapeutically, insulin differs from many other polypeptide drugs, inthe sense that overdosing may lead to severe hypoglycemia. To avoidthat, only a limited dosage of insulin can be administered each time.Such a dosage is often insufficient to maintain the basal nighttimeinsulin level needed to avoid hyperglycemia at dawn. This therapeuticdrawback can be overcome by engineering an insulin-prodrug that isbiologically inactive at the time of administration.

In this example, we aim to link insulin to HSA via a covalently stablebond. We expect such a non-dissociable molecule to share albumin'slongevity in vivo. As an extended surface area of the insulin moleculeis engaged in receptor binding (Pullen et al. 1976), we initiallythought it unlikely that such a conjugate would be biologically (andtherapeutically) active. Nevertheless, even an inactive, insulin-HSAchimera could be advantageous if the conjugate is capable of generatingthe active unmodified parent insulin with a desirable pharmacokineticpattern, Our efforts in this latter direction are brought here indetail.

Materials and Methods

(i) Materials. Human (Zn²⁺-free) insulin was donated by Novo Nordisk(Bagsvaerd, Denmark) and by Biotechnology General (Rehovot, Israel) andwas used without further purification. D-[U¹⁴-C]Glucose (4-7 mCi/mol)was obtained from DuPont-NEN (Boston, Mass.). Collagenase, type I, (134U/mg) was purchased from Worthington (Freehold, N.J.).9-Fluorenylmethoxycarbonyl-N-hydroxy-succinimide (Fmoc-OSu) anddi-tert-butyldicarbonate (t-Boc)₂O were obtained from Novabiochem(Lauifelfingen, Switzerland). MIB-NHS was obtained from Sigma-Aldrich(Rehovot, Israel). HSA was acquired from Omrix Biopharmaceutical Ltd.(Weizmann Science Park, Nes-Ziona, Israel). The protein was treated withone equivalent of dithiothretol for 20 min at pH 6.0, and thenextensively dialyzed. This treatment removes any mixed disulfide formand/or any other removable protection from cysteine-34 of HSA (36). HSAprepared by this procedure contains 0.76±0.03 mol SH per mol HSA asdetermined with DTNB (37). All other materials used in this study wereof analytical grade.

HPLC analyses were performed using a Spectra-Physics SP-8800 liquidchromatography system with an Applied Biosystems 757 variable wavelength absorbance detector and a Spectra-SYSTEM and P2000 liquidchromatography system with a Spectra-SYSTEM AS100 Autosampler andSpectra-SYSTEM UV1000, all controlled by a ThermoQuest chromatographydata system (ThermoQuest Inc., San Jose, Calif.). The column effluentswere monitored by UV absorbance at 220 nm. Analytical reverse phase-HPLCwas performed using a pre-packed Chromolith Performance RP-18e column(4.6×100 mm, Merck KGaA, Darmstadt, Germany). The column was eluted witha binary gradient established between solution A (0.1% TFA in H₂O) andsolution B (0.1% TFA in acetonitrile:H₂O; 3:1 v/v). Preparativeseparations were performed with a pre-packed Vydac, RP-18 or RP-4 column(250×22 mm, Hesperia, Calif., USA).

(ii) Biological Methods and Procedures.

Iodination of insulin and of HSA using [¹²⁵I] iodine was performed usingthe chloramine-T method (Hunter and Greenwood, 1962). Rat adipocyteswere prepared from the fat pads of male Wistar rats (100-200 g) bycollagenase digestion (Rodbel, 1964). Lipogenesis (during which [U¹⁴-C]glucose was incorporated into the lipids) was performed using theprocedure of Moody et al (Moody et al., 1974). Diabetes was induced by asingle intravenous injection of freshly prepared streptozocin (STZ)solution (55 mg/kg body wt) as previously described (Meyerovitch et al.,1987). Rats were maintained at 24° C. under conditions of controlledlighting and were fed ad libitum. Blood samples for the analysis ofblood glucose were taken from the tail veins and measured with a glucoseanalyzer (Beckman Instruments, Fullerton, Calif.) by the glucose oxidasemethod. Groups consisted of five Wistar rats weighing 170±5 g. Data arepresented as means±SE.

34(i) Pharmacological Pattern of Subcutaneously Administered HSA in theRat

¹²⁵I-labeled HSA was administered subcutaneously to rats and thepharmacological pattern was constructed as a function of time bywithdrawing blood aliquots at various time points and determining theTCA-precipitable counts for each aliquot. Subcutaneously administeredHSA is a long-lived species in the rat circulatory system in vivo. Itreached a peak value 14±1 h after administration and then declined witha t_(1/2) of 41±2 h (FIG. 24). For comparison, we subcutaneouslyadministered ¹²⁵I-insulin under similar experimental conditions. Insulinreached its peak value at 1±0.1 h and then declined with a t_(1/2) of9.0±1.0 hrs. Thus, subcutaneously administered HSA exhibits asubstantially prolonged life-time in vivo compared to insulin or anyother nonglycosylated peptide or protein of molecular mass lower than 17kDa (reviewed in Shechter et al., 2001).

34(ii) Preparation of HSA-Fmoc-Insulin

To a stirred solution of Zn²⁺ free insulin (5.9 mg, 1 μmoles in 1.0 mlof 0.1M phosphate buffer at pH 7.2) was added 505 μg (1 eq) ofMAL-Fmoc-OSu (50.5 μl from a fresh solution of MAL-Fmoc-OSu in DMF, 10mg/ml). The reaction was carried out for 20 minutes at 25° C. HSA (0.37ml from a solution of 1.6 mM) was then added to a final concentration of0.42 mM (0.6 mol/mol insulin). The reaction was carried out for 2 hrs,and the mixture was then dialyzed overnight against H₂O at 7° C.HSA-Fmoc-insulin was purified from unreacted insulin and/or frominsulin-Fmoc-MAL that had not reacted with HSA by preparative HPLC (RP-4column, Hesperia Calif. 20-100% B over 60 min with a flow rate of 10ml/min.) The fractions corresponding to HSA-Fmoc-insulin, co-eluted withHSA, were collected and lyophilized.

34(iii) Preparation of HSA-BENZ-Insulin

This (irreversible) HSA-insulin conjugate was prepared under identicalconditions to those used for HSA-Fmoc-insulin in 34(ii) above, exceptthat the heterobifunctional agent used was MIB-NHS. The reagent (32 μlfrom a fresh solution of MIB-NHS at 10 mg/ml) was reacted with insulinfor 20 min at 25° C., prior to the addition of HSA. HPLC-purifiedHSA-BENZ-insulin was characterized by MALDI-TOF MS. A mass of 72.4324kDa was found (calculated mass for 1:1 conjugate is 72.570 kDa).

34(iv) Engineering an HSA-Insulin Conjugate that Releases Insulin UnderPhysiological Conditions

As described in 34(iii) above, the covalent linking of insulin to HSAwith MIB-NHS resulted in a non-dissociable HSA-insulin conjugate havingnegligible biological potencies in vitro. We therefore theheterobifunctional agent MAL-Fmoc-OSu (Precursor 7), consisting of anFmoc-OSu derivative in which a maleimide group is attached to thefluorenyl backbone. Using MAL-Fmoc-OSu enabled us to link proteins viatheir amino side chains to the single cysteinyl residue of HSA.Previously, we have found that Fmoc moieties, linked to the amino sidechains of peptides and proteins, undergo slow hydrolysis in aqueoussolutions under physiological conditions, generating the unmodifiedparent peptides and proteins (Shechter et al., 2001; Gershonov et al.,1999).

The procedure described in detail in 34(i) above was found optimal forcoupling equimolar amounts of insulin to HSA. In brief, MAL-Fmoc-OSu isfirst reacted stoichiometrically with insulin for 20 min at pH 7.2, thisbeing a pH value at which MAL remains chemically stable for severalhours (Hazum et al., 1992). Albumin is then added with a stoichiometryof 0.6 mol per mol derivatized insulin to ensure quantitative couplingof insulin-Fmoc-MAL to the cysteinyl-34 of this carrier protein.Unreacted insulin and/or insulin-Fmoc-MAL are removed by asemi-preparative HPLC procedure.

34(v) General Features of HSA-Fmoc-Insulin

Table 6 summarizes several characteristic features of the HPLC-purifiedHSA-Fmoc-insulin we prepared. It is a highly water-soluble derivative(>200 mg/ml) in buffered near-neutral solutions (pH 6-7) owing to theextreme solubility of the carrier protein (Peters, 1996). MALDI-TOF MSanalysis revealed a molecular mass of 73.189 kDa (calculated mass forthe 1:1 conjugate is 73.250 kDa). Analytical HPLC revealed thatHSA-Fmoc-insulin co-emerges with HSA as a single symmetric peak (FIG.25; retention time 8.1 min). A molar extinction 0.1% coefficient ofε280=54,400 (ε₂₈₀ ^(0.1)=0.743) was found (calculated for 1:1 conjugateε280=62,285). The HSA-Fmoc-insulin thus obtained contains 24±3 μgcovalently linked insulin per mg HSA as judged by HPLC analysisfollowing the release of the covalently linked insulin from theconjugate by incubation at pH 10.3 for 4 hrs at 25° C. (FIG. 25).

34(vi) Biological Potency of HSA-Fmoc-Insulin

FIG. 26 shows the dose-response curve for native insulin and forHSA-Fmoc-insulin in a lipogenic assay in rat adipocytes. Based on thevalue of 24±31 g covalently linked insulin per mg HSA in the conjugate(Table 6), HSA-Fmoc-insulin has 12±3% the biological potency of thenative hormone (ED50 value=2.5±0.1 ng/ml versus ED₅₀=0.3±0.01 ng/ml forinsulin, FIG. 26). The covalent linking of insulin to HSA, using thenon-reversible agent MIB-NHS under similar experimental conditions (seeMethods), yielded a conjugate having negligible potency (ED₅₀=130±10ng/ml, being ˜0.2% of the biological potency of insulin).

34(vii) HSA-Fmoc-Insulin Releases Insulin Upon Incubation UnderPhysiological Conditions

Incubation of HSA-Fmoc-insulin at pH 10.3 for 4 hrs at 25° C. causes thecovalently linked insulin to be released quantitatively from theconjugate (FIG. 25B). In FIG. 27A, HSA-Fmoc-insulin was incubated in0.1M phosphate buffer (pH 8.5, 37° C.) and the amount of insulinreleased from the conjugate as a function of time was quantified by HPLCanalysis. At pH 8.5 the rate of Fmoc hydrolysis from Fmoc-proteinconjugates is nearly identical to that obtained in normal human serum at37° C. (Shechter et al., 2001; Gershonov et al., 1999). As shown in FIG.27A, insulin is released from the conjugate in a slow homogenousfashion, having a half-life of 24±3 hrs. After 80 hrs and 150 hrs thecumulative amount of free insulin reached 71% and 100%, respectively, ofthe initial HSA-Fmoc-insulin level.

34(viii) Reactivation of HSA-Fmoc-Insulin Upon Incubation

In FIG. 27B, aliquots were withdrawn from incubated HSA-Fmoc-insulin (pH8.5, 37° C.) at different time points and analyzed for their biologicalpotencies in a lipogenic assay in rat adipocytes. As shown in FIG. 27B,the conjugate undergoes reactivation upon incubation in a nearly linearfashion. Thus, starting from 12±3% at time 0, lipogenic potency iselevated to 31±3%, 43±4% and 59±5% following 10 hrs, 20 hrs and 40 hrsof incubation, respectively. Upon 150 hr of incubation, HSA-Fmoc-insulinregains its full biological potency (FIG. 27B).

34(ix) A Single Intraperitoneal or Subcutaneous Administration ofHSA-Fmoc-Insulin Facilitates a Prolonged Glucose-Lowering Pattern inMice

In FIG. 28, we compare the glucose-lowering pattern of HSA-Fmoc-insulin(0.4 mg/mouse, corresponding to 9.6 μg covalently linked insulin) tothat of Zn²⁺-free insulin (3 μg/mouse), following a singleadministration. For FIG. 28A, administration was intraperitoneal, whilein FIG. 28B it was subcutaneous. As shown in FIG. 28A, HSA-Fmoc-insulinfacilitates a prolonged and stable glucose-lowering pattern over aperiod of 14 hrs, exceeding by about 4 times the duration obtained bythe native hormone. Nearly the same glucose lowering patterns wereobtained following subcutaneous administration (FIG. 28B). Again, theconjugate produced a prolonged and stable glucose-lowering pattern overmany hours. A noticeable difference, however, is a delay of about 0.5hrs until the fall in glucose level following subcutaneousadministration of the conjugate commences (FIG. 28B). This suggests thatthe rate of conjugate diffusion from the subcutaneous compartment intothe circulatory system is considerably slower than that from theperitoneum. Indeed, diffusion and transportation rates of subcutaneouslyadministered proteins across capillary membranes are known to decreasein proportion with increasing size of atomic radius (Taylor and Granger,1984; Eisenberg and Crothers, 1979).

34(x) Glucose-Lowering Pattern of HSA-Fmoc-Insulin in STZ-Rats

FIG. 29 shows the glucose-lowering pattern of the conjugate after asingle subcutaneous administration in streptozocin-treated hyperglycemicrats. Here we have compared a conjugate dose (7 mg/STZ-rat) that isequipotent to the administered dose of Zn²⁺-free insulin (20 μg/STZ-rat)at the time of administration. The dosage calculation was based on 24±3μg of covalently linked insulin per mg HSA having 12% of the biologicalpotency of the free hormone (Table 6, FIG. 26). As shown in FIG. 29,HSA-Fmoc-insulin's glucose lowering effect is about 2.6 times greaterthan that of the native hormone. A small decrease is seen shortly afteradministration (i.e. at 0.5 hrs). Circulating glucose levels then fallgradually, reaching a maximal decrease at 2-3 hrs after administration(190±20 mg/dl). Hyperglycemia then reoccurs (t_(1/2=5.7) hrs). The areaunder the curve of the saline treated group, following HSA-Fmoc-insulinadministration exceeds, by about four times, comparing to that obtainedwith native hormone (integrated from FIG. 29). Thus, HSA-Fmoc-insulin isconsiderably more effective than insulin in lowering circulating glucoselevels in insulin-deficient diabetic rats.

DISCUSSION

Long fatty-acid acylated insulins that are noncovalently associated withalbumin, even with moderate affinity (Ka—10⁵M⁻¹), are long-actingspecies in vivo (Kurtzhals et al., 1995, 1996). We wondered whether thiswould be equally valid if an insulin molecule were to be covalentlylinked to the carrier protein, particularly since HSA-peptide conjugateshave been found to retain substantial parent-peptide activity (Leger etal., 2003, 2004; Holmes et al., 2000). Since the linkage of insulin toHSA through the non-reversible linker MIB-NHS yielded an inactiveconjugate (HSA-BENZ-insulin, see Example 34(iii) above), we used theFmoc-containing heterobifunctional reagent MAL-Fmoc-OSu. Thisdevelopment enabled us to link insulin (and potentially any otherpeptide or protein drug) via its amino groups to cysteine-34 of HSA. Inprevious studies we found that Fmoc moieties linked to proteins aredetached in a slow and spontaneous fashion under aqueous physiologicalconditions generating the unmodified parental proteins. The rate of Fmochydrolysis is dictated exclusively by the pH, temperature and proteinconcentration of the serum, three parameters that are maintained inmammals in strict homeostasis. We therefore anticipated thatHSA-Fmoc-insulin will yield insulin in a rather similar manner.

HSA-Fmoc-insulin is an extremely water-soluble conjugate in whichinsulin has about 12% the biological potency of the native hormone (FIG.26), in lipogenesis assay. We had expected it to have negligibleactivity, given that an extended surface area of the insulin molecule isrequired for receptor binding. This activity, however, may stem fromrelease of insulin from its conjugate during the assay, i.e. incubationfor 2 hr at 37° C. in the presence of 20 mg/ml BSA. Indeed, such anexplanation would be consistent with the inactivity of the stablecovalent conjugate HSA-BENZ-insulin. Yet, upon incubation in aqueoussolutions (i.e. pH 8.5, 37° C.) the covalently linked insulin isreleased from the HSA-Fmoc-insulin conjugate in a nearly homogenousfashion over a prolonged period (t_(1/2)=24±3 hrs) with the concomitantregeneration of insulin possessing full biological potency (FIGS. 27A,27B). HSA-Fmoc-insulin facilitates prolonged glucose lowering patternsin mice and in STZ-rats following a single subcutaneous orintraperitoneal administration. Based on administering equipotent dosesof insulin and of the conjugate at time 0, the glucose lowering potencyof HSA-Fmoc-insulin exceeds 3-4 times that facilitated by insulin interms of longevity as well as efficacy (FIGS. 28A, 28B, 29).

REFERENCES

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TABLE 1 Chemical features of MAL-FMS-NHS Characteristic Numerical valueSolubility in aqueous buffer (pH 7.2) >10 mg/ml Mass spectra data^(a)Calculated ESMS 583 Da Found ESMS for [M + H]⁺ 584.52 Da Found ESMS for[M + Na]⁺ 606.47 Da Retention time (analytical HPLC)^(b) 2.65 min Molarextinction coefficient at 280 nm^(c) 21,200 ± 200 mole⁻¹cm⁻¹ Molarextinction coefficient at 320 nm^(c) 16,100 ± 150 mole⁻¹cm⁻¹ ^(a)Massspectra were determined using the electrospray ionization technique^(b)The HPLC column used is a Chromolith column; (C₁₈); linear gradientof 10-100% of solvent B (3 ml/min). ^(c)Based on the absorbance at 280and 301 nm in PBS, pH 7.2, with compound concentration determined byquantitating the MAL-function with excess GSH and DTNB.

TABLE 2 Chemical features of PEG₄₀-FMS-IFNα2 Characteristic^(a)Numerical Value Absorbance at 280 nm^(b,c) ε₂₈₀ = 39270 ± 100 Massspectra^(d) PEG-FMS-IFNα2^(e), calculated 63569 daltonsPEG-FMS-IFNα2^(e), measured 63540 daltons Retention time (analyticalHPLC)^(f) 43 ± 0.5 min. Solubility in aqueous buffer, pH 7.4 >20 mg/ml^(a)For characterization, IFN_(α)2-FMS-MAL was dialyzed against H₂Oprior to linking PEG₄₀-SH. The final product was filtered through acentricon having a cut-off value of 50 kDa. These procedures remove freeMAL-FMS-NHS and any residual native IFN_(α)2, or IFN_(α)2-FMS-MAL thathas not been linked to PEG₄₀-SH. ^(b)Determined by UV spectroscopy.Derivative concentration was determined by acid hydrolysis of a 20 μlaliquot followed by amino acid analysis, calculated according toaspartic acid (14 residues), alanine (9 residues) and isoleucine (8residues). ^(c)Native IFN_(α)2 absorbs at 280 nm with _(ε280) = 18070(30). ^(d)Mass spectra were determined by using MALDI-TOF massspectroscopy. ^(e)Calculated mass is obtained by the additive massesfound for native IFN_(α)2 (19278 daltons); for PEG₄₀-SH (43818 daltons)and for the spacer molecule following conjugation (473 daltons).^(f)Native IFN_(α)2 elutes under identical analytical HPLC procedurewith retention time = 33.9 min.

TABLE 3 Structures and half-life time of PEG-FMS conjugates. Structure k(h⁻¹)^(b) t_(½) (h)^(c) Mal-FMS- Peptide 27^(a)

0.082 8.4 PEG₄₀₀₀₀-FMS- exendin-4^(d)

0.058 11.9 PEG₅₀₀₀-FMS- exendin-4

0.050 13.8 PEG₅₀₀₀-FMS-4- nitro- phenethylamine

0.074 9.4 PEG₄₀₀₀₀-FMS- human growth hormone (hGH)^(d)

0.059 11.8 ^(a)Rate of hydrolysis was followed by trinitrobenzenesulfonic acid assay ^(b)k is the slope constant derived from the plot ofln [PEG-FMS conjugate] against time (h) ^(c)t_(½) was determined fromthe formula t_(½) = ln2/k. ^(d)plot of hydrolysis can be found as FIG.23.

TABLE 4 Receptor binding capacity of mono- and bis-PEG-FMS-IFN-α2conjugates prior to, and following, incubation with ifnar-2-EC. MoleReceptor binding t_(1/2) of PEG₄₀- Receptor capacity followingregenerating FMS binding 50 h of incubation receptor Derivative per molecapacity^((a)) at pH 8.5, 37° C. binding designation IFNα2 % %^((b))capacity (h) Native — 100 97 ± 2 — IFNα2 (PEG₄₀- 1.0 9 ± 1  95 ± 4  9 ±1 FMS)₁- IFNα2 (PEG₄₀- 1.9 0.4 ± 0.05 92 ± 3 24 ± 3 FMS)₂- IFNα2^((a))Receptor binding capacity toward immobilized ifnar-2-EC wasassessed by the reflectometric interference spectroscopy procedure-RIFS.^((b))Incubation was performed in 0.1 M phosphate buffer pH 8.5,containing 0.5% BSA.

TABLE 5 Rate of hydrolysis of PEG₄₀-FMS-ANP at pH 8.5, 37° C.. Time ofIncubation (pH 8.5, 37° C.) hours % hydrolyzed 0 0 3 7 6 18 10 27 15 4023 50 33 75 43 87 50 100

TABLE 6 Chemophysical features of HSA-Fmoc-insulin Covalently-linkedinsulin^(a) 24 ± 3 μg/mg HSA Solubility in aqueous buffer >200 mg/ml (pH7.0) HPLC analysis retention time^(a) 8.1 min MALDI-TOF Mass SpectrumCalculated 73.250 kDa analysis^(b) (m/z) Found 73.189 kDa Molarextinction coefficient Calculated^(c) ε₂₈₀ = 62,285 Found ε₂₈₀ = 54,400ε₂₈₀ ^(0.1%) = 0.744 ^(a)Determined by HPLC analysis followingincubation of the conjugate in 0.1M Na2CO3 (pH 10.3) for 4 h at 25° C..Analytical HPLC procedures were carried out under the experimentalconditions specified in the legend to FIG. 25. Under these conditions,insulin elutes with Rt = 6.91 min and has a surface area of 187,000 ±9000 mav/μg insulin and HSA (either free or linked to insulin) eluteswith Rt = 8.1 min and has a surface area of 156,000 ± 7000 mav/μg HSA.^(b)MALDI-TOF MS analyses were carried out with theBruker-Reflex-Reflection model. ^(c)Molar extinction coefficient forHSA-Fmoc-insulin was calculated by combining the _(ε)280 values of HSA(_(ε)280 = 35,280, ref. 37), insulin (_(ε)280 = 5800) and Fmoc (_(ε)280= 10,250).

Fmoc/FMS derivative R₅—R₆-PEG PEG derivative

—NH—CO—O-PEG PEG-OCO—Cl —NH—CO—CH₂—NH₂—CO—NH-PEG PEG-NH₂ —NH—CO-PEGPEG-COOH —NH—CH₂-PEG PEG-CHO —NH—CO—NH-PEG PEG-N═C═O —NH—CS—NH-PEGPEG-N═C═S

PEG-SH

—CO—O-PEG PEG-OH —CO—NH-PEG PEG-NH₂

—NH—CO—NH-PEG PEG-NH₂

—SO₂—CH₂—CH₂—S-PEG PEG-SH

—SO₂—NH-PEG PEG-NH₂ —SO₂—O-PEG PEG-OH

—CH₂—NH-PEG PEG-NH₂

—PO₂—NH-PEG PEG-NH₂ —PO₂—O-PEG PEG-OH

—(CH₂)_(n-1)—CH₂—NH-PEG PEG-NH₂ —(CH₂)_(n-1)—CH₂—S-PEG PEG-SH

1. A compound of the formula:

wherein: R₁ is a radical of the formula:—R₅—R₆—B wherein R₂ is H or —SO₃H at position 2 of the fluorene ring; Bis maleimido, —S—CO—CH₃ or a PEG moiety; R⁵ is selected from the groupconsisting of —NH—, —S—, —CO—, —COO—, —CH₂—, —SO₂—, —SO₃—, —PO₂— and—PO₃—; and R⁶ is a bond or a radical by which the maleimido, —S—CO—CH₃or PEG moiety is attached to R₅.
 2. A compound according to claim 1,wherein: R₅ is NH—; R₆ is selected from the group consisting of —CO—,—COO—, —CH₂—, —CH(CH₃)—, CO—NH—, —CS—NH—, —CO—CH₂—NH—CO—,—CO—CH(CH₃)—NH—CO—, —CO—CH₂—NH—CO—NH,

Z is O, S or NH; R₇ is selected from the group consisting of C1-C18straight or branched alkylene, phenylene, an oxyalkylene radical having3-18 carbon atoms in the backbone, a residue of a peptide containing2-10 amino acid residues, and a residue of a saccharide containing 1-10monosaccharide residues; and R₈ is a C1-C8 straight or branchedalkylene, when B is maleimido or —S—CO—CH₃.
 3. A compound according toclaim 2 herein identified as Precursors 1-7.
 4. A compound according toclaim 2 herein identified as Precursor 8 or MAL-FMS-NHS of the formula:


5. A conjugate herein identified by the formula:(MAL-FMS)_(n)-Y wherein Y is a moiety of a peptide or protein drug, n isan integer of at least one, and Y is linked to the FMS radical throughan amino group.
 6. A conjugate according to claim 5, wherein Y isselected from the group consisting of insulin, IFN-α2, PYY₃₋₃₆,exendin-4, human growth hormone (hGH) and atrial natriuretic peptide(ANP).
 7. A compound according to claim 2, wherein R₈ is ethylene.
 8. Aconjugate according to claim 5, wherein n is 1 or 2.